G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
guanine nucleotide binding protein (G protein), q polypeptide
G00001143 (Mus musculus)

Databases (8)

Curated Gene
OTTHUMG00000020059 (Vega human gene)
ENSG00000156052 (Ensembl human gene)
2776 (Entrez Gene)
246 (G2Cdb plasticity & disease)
GNAQ (GeneCards)
600998 (OMIM)
Marker Symbol
HGNC:4390 (HGNC)
Protein Sequence
P50148 (UniProt)

Synonyms (2)

  • G-ALPHA-q
  • GAQ

Literature (125)

Pubmed - other

  • Glucagon receptor mediates calcium signaling by coupling to G alpha q/11 and G alpha i/o in HEK293 cells.

    Xu Y and Xie X

    State Key Laboratory for Drug Research, National Center for Drug Screening, Shanghai, China.

    Glucagon induces intracellular Ca(2+) ([Ca(2+)](i)) elevation by stimulating glucagon receptor (GCGR). Such [Ca(2+)](i) signaling plays important physiological roles, including glycogenolysis and glycolysis in liver cells and the survival of beta-cells. Previous studies indicated that phospholipase C (PLC) might be involved in glucagon-mediated [Ca(2+)](i) response. Other studies also debated whether cAMP accumulation mediated by GCGR/G alpha(s) coupling contributes to [Ca(2+)](i) elevation. But the exact mechanisms remain uncertain. In the present study, we found that glucagon induces [Ca(2+)](i) elevation in HEK293 cells expressing GCGR. Removing extracellular Ca(2+) did not affect glucagon-stimulated [Ca(2+)](i) response. But depleting the intracellular Ca(2+) store by thapsigargin completely inhibited glucagon-induced [Ca(2+)](i) response. Experiments with forskolin and adenylyl cyclase inhibitor revealed that cAMP is not the cause of [Ca(2+)](i) response. Further studies with G alpha(q/11) RNAi and pertussis toxin (PTX) indicated that both G alpha(q/11) and G alpha(i/o) are involved. Combination of G alpha(q/11) RNAi and G alpha(i/o) inhibition almost completely abolished glucagon-induced [Ca(2+)](i) signaling.

    Journal of receptor and signal transduction research 2009;29;6;318-25

  • Oncogenic GNAQ mutations are not correlated with disease-free survival in uveal melanoma.

    Bauer J, Kilic E, Vaarwater J, Bastian BC, Garbe C and de Klein A

    Department of Dermatology, University of Tübingen Medical Center, Liebermeisterstr. 25, Tübingen 72076, Germany. mail@j-bauer.de

    Background: Recently, oncogenic G protein alpha subunit q (GNAQ) mutations have been described in about 50% of uveal melanomas and in the blue nevi of the skin.

    Methods: GNAQ exon 5 was amplified from 75 ciliary body and choroidal melanoma DNAs and sequenced directly. GNAQ mutation status was correlated with disease-free survival (DFS), as well as other clinical and histopathological factors, and with chromosomal variations detected by FISH and CGH.

    Results: Of the 75 tumour DNA samples analysed, 40 (53.3%) harboured oncogenic mutations in GNAQ codon 209. Univariate and multivariate analysis showed that GNAQ mutation status was not significantly correlated with DFS.

    Conclusion: The GNAQ mutation status is not suitable to predict DFS. However, the high frequency of GNAQ mutations may render it a promising target for therapeutic intervention.

    British journal of cancer 2009;101;5;813-5

  • Mutational profile of GNAQQ209 in human tumors.

    Lamba S, Felicioni L, Buttitta F, Bleeker FE, Malatesta S, Corbo V, Scarpa A, Rodolfo M, Knowles M, Frattini M, Marchetti A and Bardelli A

    Laboratory of Molecular Genetics, Institute for Cancer Research and Treatment, University of Torino Medical School, University of Torino, Medical School Candiolo, Torino, Italy.

    Background: Frequent somatic mutations have recently been identified in the ras-like domain of the heterotrimeric G protein alpha-subunit (GNAQ) in blue naevi 83%, malignant blue naevi (50%) and ocular melanoma of the uvea (46%). The mutations exclusively affect codon 209 and result in GNAQ constitutive activation which, in turn, acts as a dominant oncogene.

    Methodology: To assess if the mutations are present in other tumor types we performed a systematic mutational profile of the GNAQ exon 5 in a panel of 922 neoplasms, including glioblastoma, gastrointestinal stromal tumors (GIST), acute myeloid leukemia (AML), blue naevi, skin melanoma, bladder, breast, colorectal, lung, ovarian, pancreas, and thyroid carcinomas.

    We detected the previously reported mutations in 6/13 (46%) blue naevi. Changes affecting Q209 were not found in any of the other tumors. Our data indicate that the occurrence of GNAQ mutations display a unique pattern being present in a subset of melanocytic tumors but not in malignancies of glial, epithelial and stromal origin analyzed in this study.

    PloS one 2009;4;8;e6833

  • Lack of GNAQ hotspot mutation in papillary thyroid carcinomas.

    Matsuse M, Mitsutake N, Nishihara E, Rogounovitch T, Saenko V, Rumyantsev P, Lushnikov E, Suzuki K, Miyauchi A and Yamashita S

    Thyroid : official journal of the American Thyroid Association 2009;19;8;921-2

  • Rapid changes in shear stress induce dissociation of a G alpha(q/11)-platelet endothelial cell adhesion molecule-1 complex.

    Otte LA, Bell KS, Loufrani L, Yeh JC, Melchior B, Dao DN, Stevens HY, White CR and Frangos JA

    La Jolla Bioengineering Institute, 505 Coast Blvd South, La Jolla, CA 92037, USA.

    It has been recently shown that endothelial platelet endothelial cell adhesion molecule-1 (PECAM-1) expression is pro-atherogenic. PECAM-1 is involved in sensing rapid changes in fluid shear stress but the mechanisms for activating signalling complexes at the endothelial cell junction have yet to be elucidated. Additional studies suggest the activation of membrane-bound G proteins G alpha(q/11) also mediate flow-induced responses. Here, we investigated whether PECAM-1 and G alpha(q/11) could act in unison to rapidly respond to fluid shear stress. With immunohistochemistry, we observed a co-localization of G alpha(q/11) and PECAM-1 at the cell-cell junction in the atheroprotected section of mouse aortae. In contrast, G alpha(q/11) was absent from junctions in atheroprone areas as well as in all arterial sections of PECAM-1 knockout mice. In primary human endothelial cells, temporal gradients in shear stress led to a rapid dissociation of the G alpha(q/11)-PECAM-1 complex within 30 s and a partial relocalization of the G alpha(q/11) staining to perinuclear areas within 150 min, whereas transitioning fluid flow devoid of temporal gradients did not disrupt the complex. Inhibition of G protein activation eliminated temporal gradient flow-induced G alpha(q/11)-PECAM-1 dissociation. These results allow us to conclude that G alpha(q/11)-PECAM-1 forms a mechanosensitive complex and its localization suggests the G alpha(q/11)-PECAM-1 complex is a critical mediator of vascular diseases.

    Funded by: NHLBI NIH HHS: HL-40696, R01 HL040696, R37 HL040696

    The Journal of physiology 2009;587;Pt 10;2365-73

  • Interplay between the heterotrimeric G-protein subunits Galphaq and Galphai2 sets the threshold for chemotaxis and TCR activation.

    Ngai J, Inngjerdingen M, Berge T and Taskén K

    The Biotechnology Centre of Oslo, University of Oslo, Oslo, Norway. jacob.ngai@biotek.uio.no

    Background: TCR and CXCR4-mediated signaling appears to be reciprocally regulated pathways. TCR activation dampens the chemotactic response towards the CXCR4 ligand CXCL12, while T cells exposed to CXCL12 are less prone to subsequent TCR-activation. The heterotrimeric G proteins Galphaq and Galphai2 have been implicated in CXCR4-signaling and we have recently also reported the possible involvement of Galphaq in TCR-dependent activation of Lck (Ngai et al., Eur. J. Immunol., 2008, 38: 32083218). Here we examined the role of Galphaq in migration and TCR activation.

    Results: Pre-treatment of T cells with CXCL12 led to significantly reduced Lck Y394 phosphorylation upon TCR triggering indicating heterologous desensitization. We show that knockdown of Galphaq significantly enhanced basal migration in T cells and reduced CXCL12-induced SHP-1 phosphorylation whereas Galphai2 knockdown inhibited CXCL12-induced migration.

    Conclusion: Our data suggest that Galphai2 confers migration signals in the presence of CXCL12 whereas Galphaq exerts a tonic inhibition on both basal and stimulated migrational responses. This is compatible with the notion that the level of Galphaq activation contributes to determining the commitment of the T cell either to migration or activation through the TCR.

    BMC immunology 2009;10;27

  • Angiotensin II AT2 receptor oligomers mediate G-protein dysfunction in an animal model of Alzheimer disease.

    AbdAlla S, Lother H, el Missiry A, Langer A, Sergeev P, el Faramawy Y and Quitterer U

    Heinrich-Pette-Institute, Martinistrasse 52, D-20251 Hamburg, Germany.

    Progressive neurodegeneration and decline of cognitive functions are major hallmarks of Alzheimer disease (AD). Neurodegeneration in AD correlates with dysfunction of diverse signal transduction mechanisms, such as the G-protein-stimulated phosphoinositide hydrolysis mediated by Galphaq/11. We report here that impaired Galphaq/11-stimulated signaling in brains of AD patients and mice correlated with the appearance of cross-linked oligomeric angiotensin II AT2 receptors sequestering Galphaq/11. Amyloid beta (Abeta) was causal to AT2 oligomerization, because cerebral microinjection of Abeta triggered AT2 oligomerization in the hippocampus of mice in a dose-dependent manner. Abeta induced AT2 oligomerization by a two-step process of oxidative and transglutaminase-dependent cross-linking. The induction of AT2 oligomers in a transgenic mouse model with AD-like symptoms was associated with Galphaq/11 dysfunction and enhanced neurodegeneration. Vice versa, stereotactic inhibition of AT2 oligomers by RNA interference prevented the impairment of Galphaq/11 and delayed Tau phosphorylation. Thus, Abeta induces the formation of cross-linked AT2 oligomers that contribute to the dysfunction of Galphaq/11 in an animal model of Alzheimer disease.

    The Journal of biological chemistry 2009;284;10;6554-65

  • Dominant negative AT2 receptor oligomers induce G-protein arrest and symptoms of neurodegeneration.

    AbdAlla S, Lother H, el Missiry A, Sergeev P, Langer A, el Faramawy Y and Quitterer U

    Heinrich-Pette-Institute, Martinistrasse 52, D-20251 Hamburg, Germany.

    Neurodegeneration in Alzheimer's disease (AD) correlates with dysfunction of signaling mediated by Galphaq/11. Nondissociable angiotensin II AT2 receptor oligomers are linked to the impaired Galphaq/11-stimulated signaling of AD patients and transgenic mice with AD-like symptoms. To further analyze the role of AT2 receptor oligomers, we induced the formation of AT2 oligomers in an in vitro cell system. Similarly as in vivo, sequential oxidative and transglutaminase-dependent cross-linking steps triggered the formation of AT2 oligomers in vitro. Elevated reactive oxygen species mediated oxidative cross-linking of AT2 monomers to dimers involving tyrosine residues located at putative interreceptor contact sites of the cytoplasmic loop connecting transmembrane helices III/IV. Cross-linked AT2 dimers were subsequently a substrate of activated transglutaminase-2, which targeted the carboxyl terminus of AT2 dimers, as assessed by truncated and chimeric AT2 receptors, respectively. AT2 oligomers acted as dominant negative receptors in vitro by mediating Galphaq/11 protein sequestration and Galphaq/11 protein arrest. The formation of AT2 oligomers and G-protein dysfunction could be suppressed in vitro and in vivo by an AT2 receptor mutant. Inhibition of AT2 oligomerization upon stereotactic expression of the AT2 receptor mutant revealed that Galphaq/11-sequestering AT2 oligomers enhanced the development of neurodegenerative symptoms in the hippocampus of transgenic mice with AD-like pathology. Thus, AT2 oligomers inducing Galphaq/11 arrest are causally involved in inducing symptoms of neurodegeneration.

    The Journal of biological chemistry 2009;284;10;6566-74

  • Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi.

    Van Raamsdonk CD, Bezrookove V, Green G, Bauer J, Gaugler L, O'Brien JM, Simpson EM, Barsh GS and Bastian BC

    Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia V6T1Z3, Canada.

    BRAF and NRAS are common targets for somatic mutations in benign and malignant neoplasms that arise from melanocytes situated in epithelial structures, and lead to constitutive activation of the mitogen-activated protein (MAP) kinase pathway. However, BRAF and NRAS mutations are absent in a number of other melanocytic neoplasms in which the equivalent oncogenic events are currently unknown. Here we report frequent somatic mutations in the heterotrimeric G protein alpha-subunit, GNAQ, in blue naevi (83%) and ocular melanoma of the uvea (46%). The mutations occur exclusively in codon 209 in the Ras-like domain and result in constitutive activation, turning GNAQ into a dominant acting oncogene. Our results demonstrate an alternative route to MAP kinase activation in melanocytic neoplasia, providing new opportunities for therapeutic intervention.

    Funded by: NCI NIH HHS: P01 CA025874, P01 CA025874-20A1, P01 CA025874-25A10020, P01 CA025874-280020, P01 CA025874-290020, R01 CA131524

    Nature 2009;457;7229;599-602

  • Somatic mutation of GNAQ gene is rare in common solid cancers and leukemias.

    Eom HS, Kim MS, Hur SY, Yoo NJ and Lee SH

    Acta oncologica (Stockholm, Sweden) 2009;48;7;1082-4

  • Assembly of high order G alpha q-effector complexes with RGS proteins.

    Shankaranarayanan A, Thal DM, Tesmer VM, Roman DL, Neubig RR, Kozasa T and Tesmer JJ

    Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109-2216, USA.

    Transmembrane signaling through G alpha(q)-coupled receptors is linked to physiological processes such as cardiovascular development and smooth muscle function. Recent crystallographic studies have shown how G alpha(q) interacts with two activation-dependent targets, p63RhoGEF and G protein-coupled receptor kinase 2 (GRK2). These proteins bind to the effector-binding site of G alpha(q) in a manner that does not appear to physically overlap with the site on G alpha(q) bound by regulator of G-protein signaling (RGS) proteins, which function as GTPase-activating proteins (GAPs). Herein we confirm the formation of RGS-G alpha(q)-GRK2/p63RhoGEF ternary complexes using flow cytometry protein interaction and GAP assays. RGS2 and, to a lesser extent, RGS4 are negative allosteric modulators of Galpha(q) binding to either p63RhoGEF or GRK2. Conversely, GRK2 enhances the GAP activity of RGS4 but has little effect on that of RGS2. Similar but smaller magnitude responses are induced by p63RhoGEF. The fact that GRK2 and p63RhoGEF respond similarly to these RGS proteins supports the hypothesis that GRK2 is a bona fide G alpha(q) effector. The results also suggest that signal transduction pathways initiated by GRK2, such as the phosphorylation of G protein-coupled receptors, and by p63RhoGEF, such as the activation of gene transcription, can be regulated by RGS proteins via both allosteric and GAP mechanisms.

    Funded by: NHLBI NIH HHS: HL071818, HL086865; NIDA NIH HHS: DA23252; NIGMS NIH HHS: GM076821, GM61454

    The Journal of biological chemistry 2008;283;50;34923-34

  • Oncogenic mutations in GNAQ occur early in uveal melanoma.

    Onken MD, Worley LA, Long MD, Duan S, Council ML, Bowcock AM and Harbour JW

    Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri, USA.

    Purpose: Early/initiating oncogenic mutations have been identified for many cancers, but such mutations remain unidentified in uveal melanoma (UM). An extensive search for such mutations was undertaken, focusing on the RAF/MEK/ERK pathway, which is often the target of initiating mutations in other types of cancer.

    Methods: DNA samples from primary UMs were analyzed for mutations in 24 potential oncogenes that affect the RAF/MEK/ERK pathway. For GNAQ, a stimulatory alpha(q) G-protein subunit which was recently found to be mutated in UMs, resequencing was expanded to include 67 primary UMs and 22 peripheral blood samples. GNAQ status was analyzed for association with clinical, pathologic, chromosomal, immunohistochemical, and transcriptional features.

    Results: Activating mutations at codon 209 were identified in GNAQ in 33 (49%) of 67 primary UMs, including 2 (22%) of 9 iris melanomas and 31 (54%) of 58 posterior UMs. No mutations were found in the other 23 potential oncogenes. GNAQ mutations were not found in normal blood DNA samples. Consistent with GNAQ mutation being an early or initiating event, this mutation was not associated with any clinical, pathologic, or molecular features associated with late tumor progression.

    Conclusions: GNAQ mutations occur in about half of UMs, representing the most common known oncogenic mutation in this cancer. The presence of this mutation in tumors at all stages of malignant progression suggests that it is an early event in UM. Mutations in this G-protein-coupled receptor provide new insights into UM pathogenesis and could lead to new therapeutic possibilities.

    Funded by: NEI NIH HHS: EY013169-05, P30 EY 02687, P30 EY002687, R01 EY013169, R01 EY013169-05, R01 EY1316905

    Investigative ophthalmology & visual science 2008;49;12;5230-4

  • The heterotrimeric G-protein alpha-subunit Galphaq regulates TCR-mediated immune responses through an Lck-dependent pathway.

    Ngai J, Methi T, Andressen KW, Levy FO, Torgersen KM, Vang T, Wettschureck N and Taskén K

    The Biotechnology Centre of Oslo, Nordic EMBL Partnership, University of Oslo, Oslo, Norway.

    Here, we examined the functional involvement of heterotrimeric G-proteins in TCR-induced immune responses. TCR/CD3 crosslinking resulted in activation of both Galphaq and Galphas, but not Galphai-2. Targeting of Galphas, Galphai-2 and Galphaq using siRNA demonstrated a specific role of Galphaq in TCR signaling. Jurkat TAg T cells with Galphaq knockdown displayed reduced activation of Lck and LAT phosphorylation, but paradoxically showed sustained ERK1/2 phosphorylation and increased NFAT-AP-1-reporter activity implicating Galphaq in the negative control of downstream signaling and IL-2-promoter activity. Primary T cells isolated from Galphaq-deficient mice had a similar TCR signaling response with reduced proximal LAT phosphorylation, sustained ERK1/2 phosphorylation and augmented immune responses including increased secretion of IL-2, IL-5, IL-12 and TNF-alpha. The effects on NFAT-AP-1-reporter activity were sensitive to the Src family kinase inhibitor PP2 and were reversed by transient expression of constitutively active Lck. Furthermore, expression of constitutively active Galphaq Q209L elevated Lck activity and Zap-70 phosphorylation. Together these data argue for a role of Galphaq in the fine-tuning of proximal TCR signals at the level of Lck and a negative regulatory role of Galphaq in transcriptional activation of cytokine responses.

    European journal of immunology 2008;38;11;3208-18

  • Phorbol 12-myristate 13-acetate (PMA) responsive sequence in Galphaq promoter during megakaryocytic differentiation. Regulation by EGR-1 and MAP kinase pathway.

    Jalagadugula G, Dhanasekaran DN and Rao AK

    Sol Sherry Thrombosis Research Center, Temple University School of Medicine, 3400 N. Broad St., OMS-300, Philadelphia, PA 19140, USA.

    Galphaq plays a major role in platelet signal transduction, but little is known regarding its transcriptional regulation. We have reported that Galphaq is upregulated during phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic transformation of human erythroleukemia (HEL) cells and regulated by EGR-1, an early growth transcription factor. These studies focused on the initial 238 bp of the 5' upstream region of the Galphaq gene. In the present studies we characterize a minimal region -1042/-1037 bp from ATG in the 5' upstream of the Galphaq promoter that is associated with PMA responsiveness. In luciferase reporter gene studies in HEL cells, Galphaq 5' upstream promoter sequence -1042/-1 showed an about four-fold increased activity in PMA-treated compared to untreated cells. Deletion of 6-nt -1042/-1037 eliminated the difference. Gel-shift studies on Galphaq probe (-1042/-1012 bp) revealed binding of EGR-1 with PMA-treated but not untreated nuclear extracts, and this was dependent on the sequence -1042/-1037. Silencing of endogenous EGR-1 inhibited Galphaq induction by PMA. MEK/ERK inhibitor U0126 blocked PMA effect on promoter activity of the -1042/-1 construct. In conclusion, EGR-1 binding to sequence -1042/-1037 bp in Galphaq promoter mediates the induction of Galphaq gene by PMA via the MEK/ERK signaling pathway. These studies provide the first evidence of a PMA-responsive element in Galphaq promoter, and new insights into regulation of Galphaq gene by EGR-1.

    Funded by: NHLBI NIH HHS: HL056724-08, R01 HL056724, R01 HL056724-08, R01 HL56724

    Thrombosis and haemostasis 2008;100;5;821-8

  • MLK3 limits activated Galphaq signaling to Rho by binding to p63RhoGEF.

    Swenson-Fields KI, Sandquist JC, Rossol-Allison J, Blat IC, Wennerberg K, Burridge K and Means AR

    Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA.

    Mixed lineage kinase 3 (MLK3) is a MAP3K that activates the JNK-dependent MAPK pathways. Here, we show that MLK3 is required for cell migration in a manner independent of its role as a MAP3K or MLK3 kinase activity. Rather, MLK3 functions in a regulated way to limit levels of the activated GTPase Rho by binding to the Rho activator, p63RhoGEF/GEFT, which, in turn, prevents its activation by Galphaq. These findings demonstrate a scaffolding role for MLK3 in controlling the extent of Rho activation that modulates cell migration. Moreover, they suggest that MLK3 functions as a network hub that links a number of signaling pathways.

    Funded by: NCI NIH HHS: CA082845-09, P30 CA014236, R01 CA082845, R01 CA082845-09; NIGMS NIH HHS: GM29860

    Molecular cell 2008;32;1;43-56

  • Induction of the Galpha(q) signaling cascade by the human immunodeficiency virus envelope is required for virus entry.

    Harmon B and Ratner L

    Division of Molecular Oncology, Washington University School of Medicine, St. Louis, Missouri, USA.

    Binding of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) with the primary receptor CD4 and one of two coreceptors, CXCR4 or CCR5, activates a signaling cascade resulting in Rac-1 GTPase activation and stimulation of actin cytoskeletal reorganizations critical for HIV-1-mediated membrane fusion. The mechanism by which HIV-1 Env induces Rac-1 activation and subsequent actin cytoskeleton rearrangement is unknown. In this study, we show that Env-mediated Rac-1 activation is dependent on the activation of Galpha(q) and its downstream targets. Fusion and Rac-1 activation are mediated by Galpha(q) and phospholipase C (PLC), as shown by attenuation of fusion and Rac-1 activation in cells either expressing small interfering RNA (siRNA) targeting Galpha(q) or treated with the PLC inhibitor U73122. Rac-1 activation and fusion were also blocked by multiple protein kinase C inhibitors, by inhibitors of intracellular Ca2+ release, by Pyk2-targeted siRNA, and by the Ras inhibitor S-trans,trans-farnesylthiosalicylic acid (FTS). Fusion was blocked without altering cell viability or cell surface localization of CD4 and CCR5. Similar results were obtained when cell fusion was induced by Env expressed on viral and cellular membranes and when cell lines or primary cells were the target. Treatment with inhibitors and siRNA specific for Galpha(i) or Galpha(s) signaling mediators had no effect on Env-mediated Rac-1 activation or cell fusion, indicating that the Galpha(q) pathway alone is responsible. These results could provide a new focus for therapeutic intervention with drugs targeting host signaling mediators rather than viral molecules, a strategy which is less likely to result in resistance.

    Funded by: NIAID NIH HHS: AI24745, R01 AI024745, T32 AI007172

    Journal of virology 2008;82;18;9191-205

  • Regulation of G protein-coupled receptor activities by the platelet-endothelial cell adhesion molecule, PECAM-1.

    Yeh JC, Otte LA and Frangos JA

    La Jolla Bioengineering Institute, 505 Coast Boulevard South, Suite 406, La Jolla, California 92037, USA.

    It is becoming increasingly evident that the cell-cell junction is a major signaling center. Here we show that the Galphaq/11 subunit of heterotrimeric G proteins forms a complex with platelet-endothelial cell adhesion molecule 1 (PECAM-1), a junctional protein that has been shown to be involved in mechanosignaling in endothelial cells. To understand the role of PECAM-1 in this complex, we determined the critical regions of PECAM-1 involved in this interaction. By expressing truncated forms of PECAM-1 in human embryonic kidney (HEK293) cells, we found that the cytoplasmic domain of PECAM-1 is not required for its association with Galphaq/11. Domain swapping of PECAM-1 with intracellular cell adhesion molecule 1 (ICAM-1), a protein that does not form a complex with Galphaq/11, provides evidence that the extracellular domain of PECAM-1 is critical for this interaction. This result also suggests that PECAM-1 does not directly interact with Galphaq/11. Coexpression of bradykinin receptor B2 (BKRB2), a Galphaq/11-coupled receptor, with PECAM-1 enhances formation of the PECAM-1-Galphaq/11 complex, suggesting an interaction between PECAM-1 and BKRB2. Co-immunoprecipitation experiments indicate that these two molecules indeed form a complex when expressed in HEK293 cells. Activation of ERK1/2 by bradykinin in HUVEC is enhanced when PECAM-1 expression is inhibited by transfection of small interference RNA against PECAM-1. Taken together, our results provide evidence of interaction of PECAM-1 with BKRB2 and of its possible role in regulating G protein-coupled receptor (GPCR) and G protein functions.

    Funded by: NHLBI NIH HHS: HL40696, R01 HL040696

    Biochemistry 2008;47;34;9029-39

  • G-protein-dependent and -independent pathways regulate proteinase-activated receptor-2 mediated p65 NFkappaB serine 536 phosphorylation in human keratinocytes.

    Goon Goh F, Sloss CM, Cunningham MR, Nilsson M, Cadalbert L and Plevin R

    Strathclyde Institute of Pharmacy and Biomedical Sciences, Division of Physiology and Pharmacology, University of Strathclyde, 27 Taylor Street, Glasgow, G4 0NR, Scotland, UK.

    The mechanisms underpinning the coupling of GPCRs, such as PAR-2, to the phosphorylation of p65 NFkappaB have not been investigated. In the current study we found that trypsin and the selective PAR-2 activating peptide, 2f-LIGKV-OH, stimulated large and sustained increases in the serine 536 phosphorylation of p65/RelA in a transfected skin epithelial cell line and primary keratinocytes. Parallel experiments showed that in both cell types, p65 NFkappaB phosphorylation is mediated through the selective activation of IKK2. Treatment with PKC inhibitor GF109203X or PKCalpha siRNA reduced phosphorylation at 15 min but not 30 min, whilst rottlerin, a selective PKCdelta inhibitor and PKCdelta siRNA reduced the response at both time points. Pre-treatment of cells with the novel Gq/11 inhibitor YM-254890 and Gq/11 siRNA caused a similar pattern of inhibition and also reduced PAR-2-mediated NFkappaB transcriptional activity. Furthermore, stimulation of cells through a novel PAR-2 mutant PAR-2(34-43), delayed p65 phosphorylation but was without effect on the kinetics of ERK activation. Inhibition of Gi or G12/13 pathways by pertussis toxin pre-treatment or over-expression of the RGS mutant Lsc, also did not effect NFkappaB phosphorylation. Taken together these data indicate dependency for Gq/11 in early phosphorylation of p65 NFkappaB and this subsequently affects initial NFkappaB-dependent gene transcriptional activity, however later regulation of p65 is unaffected. Overall these novel data demonstrate an IKK2-dependent, predominantly G-protein-independent pathway involved in PAR-2 regulation of NFkappaB phosphorylation in keratinocytes.

    Cellular signalling 2008;20;7;1267-74

  • Reviews in molecular biology and biotechnology: transmembrane signaling by G protein-coupled receptors.

    Luttrell LM

    Division of Endocrinology, Diabetes and Medical Genetics, Department of Medicine, Medical University of South Carolina, 96 Jonathan Lucas Street, 816 CSB, P.O. Box 250624, Charleston, SC 29425, USA. luttrell@musc.edu

    As the most diverse type of cell surface receptor, the importance heptahelical G protein-coupled receptors (GPCRs) to clinical medicine cannot be overestimated. Visual, olfactory and gustatory sensation, intermediary metabolism, cell growth and differentiation are all influenced by GPCR signals. The basic receptor-G protein-effector mechanism of GPCR signaling is tuned by a complex interplay of positive and negative regulatory events that amplify the effect of a hormone binding the receptor or that dampen cellular responsiveness. The association of heptahelical receptors with a variety of intracellular partners other than G proteins has led to the discovery of potential mechanisms of GPCR signaling that extend beyond the classical paradigms. While the physiologic relevance of many of these novel mechanisms of GPCR signaling remains to be established, their existence suggests that the mechanisms of GPCR signaling are even more diverse than previously imagined.

    Molecular biotechnology 2008;39;3;239-64

  • Dissociation of heterotrimeric g proteins in cells.

    Lambert NA

    Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, GA 30912-2300, USA. nlambert@mcg.edu

    Heterotrimeric G proteins dissociate into their component Galpha and Gbetagamma subunits when these proteins are activated in solution. Until recently, it has not been known if subunit dissociation also occurs in cells. The development of optical methods to study G protein activation in live cells has made it possible to demonstrate heterotrimer dissociation at the plasma membrane. However, subunit dissociation is far from complete, and many active [guanosine triphosphate (GTP)-bound] heterotrimers are intact in a steady state. This unexpectedly reluctant dissociation calls for inclusion of a GTP-bound heterotrimeric state in models of the G protein cycle and places renewed emphasis on the relation between subunit dissociation and effector activation.

    Science signaling 2008;1;25;re5

  • Pharmacological assessment of m1 muscarinic acetylcholine receptor-gq/11 protein coupling in membranes prepared from postmortem human brain tissue.

    Salah-Uddin H, Thomas DR, Davies CH, Hagan JJ, Wood MD, Watson JM and Challiss RA

    Department of Cell Physiology and Pharmacology, University of Leicester, Henry Wellcome Building, Lancaster Road, Leicester LE1 9HN, United Kingdom.

    Using a selective Galpha(q/11) protein antibody capture guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding approach, it has been possible to perform a quantitative pharmacological examination of the functional activity of the M(1) muscarinic acetylcholine receptor (mAChR) in membranes prepared from human postmortem cerebral cortex. Oxotremorine-M caused a > or = 2-fold increase in [35S]GTPgammaS-Galpha(q/11) binding with a pEC(50) of 6.06 +/- 0.16 in Brodmann's areas 23 and 25 that was almost completely inhibited by preincubation of membranes with the M(1) mAChR subtype-selective antagonist muscarinic toxin-7. In addition, the orthosteric and allosteric agonists, xanomeline [3(3-hexyloxy-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine] and AC-42 (4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine hydrogen chloride), increased [35S]-GTPgammaS-Galpha(q/11) binding, but with reduced intrinsic activities, inducing maximal responses that were 42 +/- 1 and 44 +/- 2% of the oxotremorine-M-induced response, respectively. These data indicate that the M(1) receptor is the predominant mAChR subtype coupling to the Galpha(q/11) G protein in these brain regions and that it is possible to quantify the potency and intrinsic activity of full and partial M(1) mAChR receptor agonists in postmortem human brain using a selective Galpha(q/11) protein antibody capture [35S]GTPgammaS binding assay.

    The Journal of pharmacology and experimental therapeutics 2008;325;3;869-74

  • Phosphodiesterase 8B gene variants are associated with serum TSH levels and thyroid function.

    Arnaud-Lopez L, Usala G, Ceresini G, Mitchell BD, Pilia MG, Piras MG, Sestu N, Maschio A, Busonero F, Albai G, Dei M, Lai S, Mulas A, Crisponi L, Tanaka T, Bandinelli S, Guralnik JM, Loi A, Balaci L, Sole G, Prinzis A, Mariotti S, Shuldiner AR, Cao A, Schlessinger D, Uda M, Abecasis GR, Nagaraja R, Sanna S and Naitza S

    Laboratory of Genetics, National Institute on Aging, Baltimore, MD 21224, USA.

    Thyroid-stimulating hormone (TSH) controls thyroid growth and hormone secretion through binding to its G protein-coupled receptor (TSHR) and production of cyclic AMP (cAMP). Serum TSH is a sensitive indicator of thyroid function, and overt abnormalities in thyroid function lead to common endocrine disorders affecting approximately 10% of individuals over a life span. By genotyping 362,129 SNPs in 4,300 Sardinians, we identified a strong association (p = 1.3 x 10(-11)) between alleles of rs4704397 and circulating TSH levels; each additional copy of the minor A allele was associated with an increase of 0.13 muIU/ml in TSH. The single-nucleotide polymorphism (SNP) is located in intron 1 of PDE8B, encoding a high-affinity cAMP-specific phosphodiesterase. The association was replicated in 4,158 individuals, including additional Sardinians and two genetically distant cohorts from Tuscany and the Old Order Amish (overall p value = 1.9 x 10(-20)). In addition to association of TSH levels with SNPs in PDE8B, our genome scan provided evidence for association with PDE10A and several biologically interesting candidates in a focused analysis of 24 genes. In particular, we found evidence for association of TSH levels with SNPs in the THRB (rs1505287, p = 7.3 x 10(-5)), GNAQ (rs10512065, p = 2.0 x 10(-4)), TG (rs2252696, p = 2.2 x 10(-3)), POU1F1 (rs1976324, p = 3.9 x 10(-3)), PDE4D (rs27178, p = 8.3 x 10(-3)), and TSHR (rs4903957, p = 8.6 x 10(-3)) loci. Overall, the results suggest a primary effect of PDE8B variants on cAMP levels in the thyroid. This would affect production of T4 and T3 and feedback to alter TSH release by the pituitary. PDE8B may thus provide a candidate target for the treatment of thyroid dysfunction.

    Funded by: Intramural NIH HHS; NCRR NIH HHS: M01 RR 000052, M01 RR000052, M01 RR016500; NHGRI NIH HHS: HG002651, R01 HG002651; NHLBI NIH HHS: HL084729, U01 HL072515, U01 HL084729, U01 HL72515; NIA NIH HHS: N01-AG-1-2109, N01-AG-821336, N01-AG-916413, R01 AG018728, R01 AG18728; NIMHD NIH HHS: 263 MD 821336, 263 MD 916413, R01 MD009164; PHS HHS: 263-MA-410953

    American journal of human genetics 2008;82;6;1270-80

  • IGF-II/mannose-6-phosphate receptor signaling induced cell hypertrophy and atrial natriuretic peptide/BNP expression via Galphaq interaction and protein kinase C-alpha/CaMKII activation in H9c2 cardiomyoblast cells.

    Chu CH, Tzang BS, Chen LM, Kuo CH, Cheng YC, Chen LY, Tsai FJ, Tsai CH, Kuo WW and Huang CY

    Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung 402, Taiwan, ROC.

    The role played by IGF-II in signal transduction through the IGF-II/mannose-6-phosphate receptor (IGF2R) in heart tissue has been poorly understood. In our previous studies, we detected an increased expression of IGF-II and IGF2R in cardiomyocytes that had undergone pathological hypertrophy. We hypothesized that after binding with IGF-II, IGF2R may trigger intracellular signaling cascades involved in the progression of pathologically cardiac hypertrophy. In this study, we used immunohistochemical analysis of the human cardiovascular tissue array to detect expression of IGF2R. In our study of H9c2 cardiomyoblast cell cultures, we used the rhodamine phalloidin staining to measure the cell hypertrophy and western blot to measure the expression of cardiac hypertrophy markers atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in cells treated with IGF-II. We found that a significant association between IGF2R overexpression and myocardial infarction. The treatment of H9c2 cardiomyoblast cells with IGF-II not only induced cell hypertrophy but also increased the protein level of ANP and BNP. Using Leu27IGF-II, an analog of IGF-II which interacts selectively with the IGF2R, to specifically activate IGF2R signaling cascades, we found that binding of Leu27IGF-II to IGF2R led to an increase in the phosphorylation of protein Kinase C (PKC)-alpha and calcium/calmodulin-dependent protein kinase II (CaMKII) in a Galphaq-dependent manner. By the inhibition of PKC-alpha/CaMKII activity, we found that IGF-II and Leu27IGF-II-induced cell hypertrophy and upregulation of ANP and BNP were significantly suppressed. Taken together, this study provides a new insight into the effects of the IGF2R and its downstream signaling in cardiac hypertrophy. The suppression of IGF2R signaling pathways may be a good strategy to prevent the progression of pathological hypertrophy.

    The Journal of endocrinology 2008;197;2;381-90

  • Characterization of the GNAQ promoter and association of increased Gq expression with cardiac hypertrophy in humans.

    Frey UH, Lieb W, Erdmann J, Savidou D, Heusch G, Leineweber K, Jakob H, Hense HW, Löwel H, Brockmeyer NH, Schunkert H and Siffert W

    Institute of Pharmacogenetics, D-45122 Essen, Germany. ulrich.frey@uk-essen.de

    Aims: Transgenic mice with cardiac overexpression of Gq develop cardiac hypertrophy, apoptosis, and heart failure. Similar mechanisms may contribute to human left ventricular hypertrophy (LVH). However, mechanisms regulating transcription of the human GNAQ gene encoding the Gq protein are unknown and single-nucleotide polymorphisms have not been reported.

    We delineated essential elements for transcription in the human GNAQ promoter using reporter assays and showed promoter induction by serum and angiotensin II. Sequencing of the whole promoter revealed a common (minor allele frequency 0.48) dinucleotide polymorphism at position -694/-695, resulting in an exchange of two adjacent nucleotides (TT > GC). The GC allele had increased transcription factor binding and was associated with enhanced transcriptional activation by serum or angiotensin II, resulting in enhanced Gq expression and intracellular signalling. Genotyping a population-based survey (n = 1204) revealed a higher prevalence of LVH in individuals with the GC/GC genotype [odds ratio (OR) 4.07; 95% CI 1.63-10.16; P = 0.003], this effect being more pronounced in women (OR 5.52; P = 0.005).

    Conclusion: A novel polymorphism in the Gq promoter region is associated with enhanced promoter activity, Gq expression, intracellular signal transduction, and increased prevalence of LVH, particularly in women.

    European heart journal 2008;29;7;888-97

  • The time-course of agonist-induced solubilization of trimeric G(q)alpha/G(11)alpha proteins resolved by two-dimensional electrophoresis.

    Durchánková D, Novotný J and Svoboda P

    Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.

    Prolonged agonist stimulation results in specific transfer of activated Galpha subunits of G(q)alpha/G(11)alpha family from particulate membrane fraction to soluble (cytosol) cell fraction isolated as 250,000 x g supernatant. In this study, we have used 2D electrophoresis for more defined resolution of Galpha subunits of G(q)alpha/G(11)alpha family and followed the time course of solubilization effect. The small signal of soluble G proteins was already detected in control, hormone-unexposed cells. Hormone stimulation resulted in a slow but continuous increase of both intensity and number of immunoreactive signals/spots of these G proteins (10, 30, 60, 120 and 240 min). At longer times of agonist exposure (>2 hours), a marked increase of G(q)alpha/G(11)alpha proteins was detected. The maximal level of soluble G(q)alpha/G(11)alpha proteins was reached after 16 hours of continuous agonist exposure. At this time interval, eight individual immunoreactive signals of G(q)alpha/G(11)alpha proteins could be resolved. The relative proportion among these spots was 15:42:10:11:7:7:2:5. Solubilization of this class of Galpha proteins was thus observed after prolonged agonist stimulation only, induced by ultra high concentration of hormone and in cells expressing a large number of GPCRs. Our data therefore rather indicate tight/persisting binding of G(q)alpha/G(11)alpha proteins to the membrane.

    Physiological research 2008;57;2;195-203

  • Structure of Galphaq-p63RhoGEF-RhoA complex reveals a pathway for the activation of RhoA by GPCRs.

    Lutz S, Shankaranarayanan A, Coco C, Ridilla M, Nance MR, Vettel C, Baltus D, Evelyn CR, Neubig RR, Wieland T and Tesmer JJ

    Institute of Experimental and Clinical Pharmacology and Toxicology, Medical Faculty Mannheim, University of Heidelberg, Maybachstrasse 14, D-68169 Mannheim, Germany.

    The guanine nucleotide exchange factor p63RhoGEF is an effector of the heterotrimeric guanine nucleotide-binding protein (G protein) Galphaq and thereby links Galphaq-coupled receptors (GPCRs) to the activation of the small-molecular-weight G protein RhoA. We determined the crystal structure of the Galphaq-p63RhoGEF-RhoA complex, detailing the interactions of Galphaq with the Dbl and pleckstrin homology (DH and PH) domains of p63RhoGEF. These interactions involve the effector-binding site and the C-terminal region of Galphaq and appear to relieve autoinhibition of the catalytic DH domain by the PH domain. Trio, Duet, and p63RhoGEF are shown to constitute a family of Galphaq effectors that appear to activate RhoA both in vitro and in intact cells. We propose that this structure represents the crux of an ancient signal transduction pathway that is expected to be important in an array of physiological processes.

    Funded by: NCI NIH HHS: Y1-CO-1020; NHLBI NIH HHS: HL071818, HL086865; NIGMS NIH HHS: Y1-GM-1104

    Science (New York, N.Y.) 2007;318;5858;1923-7

  • A functional polymorphism of the Galphaq (GNAQ) gene is associated with accelerated mortality in African-American heart failure.

    Liggett SB, Kelly RJ, Parekh RR, Matkovich SJ, Benner BJ, Hahn HS, Syed FM, Galvez AS, Case KL, McGuire N, Odley AM, Sparks L, Kardia SL and Dorn GW

    Department of Medicine, Cardiopulmonary Genomics Program, University of Maryland, Baltimore, MD, USA.

    Galphaq, encoded by the human GNAQ gene, is an effector subunit of the Gq heterotrimeric G-protein and the convergence point for signaling of multiple Gq-coupled neurohormonal receptors. To identify naturally occurring mutations that could modify GNAQ transcription, we examined genomic DNA isolated from 355 normal subjects for genetic variants in transcription factor binding motifs. Of seven variants identified, the most common was a GC to TT dinucleotide substitution at -694/-695 (allele frequency of 0.467 in Caucasians and 0.329 in African Americans) within a GC-rich domain containing consensus binding sites for Sp-1, c-rel and EGR-1. In promoter-reporter analyses, the TT substitution increased promoter activity in cultured neonatal rat cardiac myocytes and human HEK fibroblasts by approximately 30% at baseline and after stimulation with phorbol ester. Two other relatively common polymorphisms, -173G/A and -168G/A, did not affect promoter activity. Since altered expression/activity of Galphaq is implicated in heart disease, we re-sequenced the GNAQ promoter in 1052 prospectively followed heart failure patients. The TT variant was not increased in heart failure, but was associated with decreased survival time among African Americans, with an adjusted RR of death/cardiac transplant of 1.95 (95% CI = 1.21-3.13) for heterozygotes and 2.4 (95% CI = 1.36-4.26) for homozygotes. Gel mobility shift assays showed that this GC/TT substitution eliminated Sp-1 binding without affecting c-rel or EGR-1 binding to this promoter fragment. Thus, the GNAQ -694/-695 promoter polymorphism alters transcription factor binding, increases promoter activity and adversely affects outcome in human heart failure.

    Funded by: NHLBI NIH HHS: P50 HL77101

    Human molecular genetics 2007;16;22;2740-50

  • Galphaq directly activates p63RhoGEF and Trio via a conserved extension of the Dbl homology-associated pleckstrin homology domain.

    Rojas RJ, Yohe ME, Gershburg S, Kawano T, Kozasa T and Sondek J

    Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599, USA.

    The coordinated cross-talk from heterotrimeric G proteins to Rho GTPases is essential during a variety of physiological processes. Emerging data suggest that members of the Galpha(12/13) and Galpha(q/11) families of heterotrimeric G proteins signal downstream to RhoA via distinct pathways. Although studies have elucidated mechanisms governing Galpha(12/13)-mediated RhoA activation, proteins that functionally couple Galpha(q/11) to RhoA activation have remained elusive. Recently, the Dbl-family guanine nucleotide exchange factor (GEF) p63RhoGEF/GEFT has been described as a novel mediator of Galpha(q/11) signaling to RhoA based on its ability to synergize with Galpha(q/11) resulting in enhanced RhoA signaling in cells. We have used biochemical/biophysical approaches with purified protein components to better understand the mechanism by which activated Galpha(q) directly engages and stimulates p63RhoGEF. Basally, p63RhoGEF is autoinhibited by the Dbl homology (DH)-associated pleckstrin homology (PH) domain; activated Galpha(q) relieves this autoinhibition by interacting with a highly conserved C-terminal extension of the PH domain. This unique extension is conserved in the related Dbl-family members Trio and Kalirin and we show that the C-terminal Rho-specific DH-PH cassette of Trio is similarly activated by Galpha(q).

    Funded by: NIGMS NIH HHS: P01 GM065533, P01-GM65533, R01 GM062299, R01 GM062299-05A1, R01 GM6145408, R01-GM62299

    The Journal of biological chemistry 2007;282;40;29201-10

  • Selective inhibition of Cav3.3 T-type calcium channels by Galphaq/11-coupled muscarinic acetylcholine receptors.

    Hildebrand ME, David LS, Hamid J, Mulatz K, Garcia E, Zamponi GW and Snutch TP

    Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4.

    T-type calcium channels play critical roles in controlling neuronal excitability, including the generation of complex spiking patterns and the modulation of synaptic plasticity, although the mechanisms and extent to which T-type Ca(2+) channels are modulated by G-protein-coupled receptors (GPCRs) remain largely unexplored. To examine specific interactions between T-type Ca(2+) channel subtypes and muscarinic acetylcholine receptors (mAChRS), the Cav3.1 (alpha(1G)), Cav3.2 (alpha(1H)), and Cav3.3 (alpha) T-type Ca(2+)(1I)channels were co-expressed with the M1 Galpha(q/11)-coupled mAChR. Perforated patch recordings demonstrate that activation of M1 receptors has a strong inhibitory effect on Cav3.3 T-type Ca(2+) currents but either no effect or a moderate stimulating effect on Cav3.1 and Cav3.2 peak current amplitudes. This differential modulation was observed for both rat and human T-type Ca(2+) channel variants. The inhibition of Cav3.3 channels by M1 receptors is reversible, use-independent, and associated with a concomitant increase in inactivation kinetics. Loss-of-function experiments with genetically encoded antagonists of Galpha and Gbetagamma proteins and gain-of-function experiments with genetically encoded Galpha subtypes indicate that M1 receptor-mediated inhibition of Cav3.3 occurs through Galpha(q/11). This is supported by experiments showing that activation of the M3 and M5 Galpha(q/11)-coupled mAChRs also causes inhibition of Cav3.3 currents, although Galpha(i)-coupled mAChRs (M2 and M4) have no effect. Examining Cav3.1-Cav3.3 chimeric channels demonstrates that two distinct regions of the Cav3.3 channel are necessary and sufficient for complete M1 receptor-mediated channel inhibition and represent novel sites not previously implicated in T-type channel modulation.

    The Journal of biological chemistry 2007;282;29;21043-55

  • The lipid raft proteins flotillins/reggies interact with Galphaq and are involved in Gq-mediated p38 mitogen-activated protein kinase activation through tyrosine kinase.

    Sugawara Y, Nishii H, Takahashi T, Yamauchi J, Mizuno N, Tago K and Itoh H

    Laboratory of Signal Transduction, Department of Cell Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.

    The heterotrimeric G protein alpha q subunit (Galphaq) mediates a variety of cell functions by activating the effector molecule phospholipase Cbeta. Galphaq activity is regulated by G protein betagamma subunits, G protein-coupled receptors, RGS proteins, and Ric-8. In this study, we identified the lipid raft resident proteins, flotillin-1/reggie-2 and flotillin-2/reggie-1, as Galphaq-binding proteins. The interactions of Galphaq and flotillins were independent of the nucleotide-binding state of Galphaq, and the N-terminal portion of flotillins was critical for the interaction. A short interfering RNA-mediated knockdown of flotillins, particularly flotillin-2, attenuated the UTP-induced activation of p38 mitogen-activated protein kinase (MAPK) but not that of ERK1/2. The activation of p38 MAPK was inhibited by the Src family tyrosine kinase inhibitor PP2 and the cholesterol-depleting agent methyl-beta-cyclodextrin, which is generally used for the disruption of lipid rafts. In contrast, the activation of ERK1/2 was not inhibited by these compounds. These lines of evidence suggested that a Gq-coupled receptor activates specifically p38 MAPK through lipid rafts and Src kinase activation, in which flotillins positively modulate the Gq signaling.

    Cellular signalling 2007;19;6;1301-8

  • Galphaq-TRPC6-mediated Ca2+ entry induces RhoA activation and resultant endothelial cell shape change in response to thrombin.

    Singh I, Knezevic N, Ahmmed GU, Kini V, Malik AB and Mehta D

    Department of Pharmacology and Center for Lung and Vascular Biology, College of Medicine, University of Illinois, Chicago, Illinois 60612, USA.

    RhoA activation and increased intracellular Ca(2+) concentration mediated by the activation of transient receptor potential channels (TRPC) both contribute to the thrombin-induced increase in endothelial cell contraction, cell shape change, and consequently to the mechanism of increased endothelial permeability. Herein, we addressed the possibility that TRPC signals RhoA activation and thereby contributes in actinomyosin-mediated endothelial cell contraction and increased endothelial permeability. Transduction of a constitutively active Galphaq mutant in human pulmonary arterial endothelial cells induced RhoA activity. Preventing the increase in intracellular Ca2+ concentration by the inhibitor of Galphaq or phospholipase C and the Ca2+ chelator, BAPTA-AM, abrogated thrombin-induced RhoA activation. Depletion of extracellular Ca2+ also inhibited RhoA activation, indicating the requirement of Ca2+ entry in the response. RhoA activation could not be ascribed to storeoperated Ca2+ (SOC) entry because SOC entry induced with thapsigargin or small interfering RNA-mediated inhibition of TRPC1 expression, the predominant SOC channel in these endothelial cells, failed to alter RhoA activity. However, activation of receptor-operated Ca2+ entry by oleoyl-2-acetyl-sn-glycerol, the membrane permeable analogue of the Galphaq-phospholipase C product diacylglycerol, induced RhoA activity. Receptor-operated Ca2+ activation was mediated by TRPC6 because small interfering RNA-induced TRPC6 knockdown significantly reduced Ca2+ entry. TRPC6 knockdown also prevented RhoA activation, myosin light chain phosphorylation, and actin stress fiber formation as well as inter-endothelial junctional gap formation in response to either oleoyl-2-acetyl-sn-glycerol or thrombin. TRPC6-mediated RhoA activity was shown to be dependent on PKCalpha activation. Our results demonstrate that Galphaq activation of TRPC6 signals the activation of PKCalpha, and thereby induces RhoA activity and endothelial cell contraction.

    Funded by: NHLBI NIH HHS: HL 45638, R01 HL071794, R01 HL084153; PHS HHS: 084153, 71794

    The Journal of biological chemistry 2007;282;11;7833-43

  • Early growth response transcription factor EGR-1 regulates Galphaq gene in megakaryocytic cells.

    Jalagadugula G, Dhanasekaran DN, Kim S, Kunapuli SP and Rao AK

    Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140, USA.

    Background: Galphaq (Gene GNAQ) plays a major role in platelet signal transduction but little is known regarding its transcriptional regulation.

    Objectives: We studied Galphaq promoter activity using luciferase reporter gene assays in human erythroleukemia (HEL) cells treated with phorbol 12-myristate 13-acetate (PMA) for 24 h to induce megakaryocytic transformation.

    PMA-treated HEL cells showed enhanced Galphaq expression. Reporter (luciferase) gene studies on 5' upstream construct (up to -116 bp from ATG) revealed a negative regulatory site at -238/-202 and two positive sites at -203/-138 and -1116/-731. The positive regulatory region -203/-138 contained overlapping Sp1/AP-2/EGR-1 consensus sites. Gel shift studies on Galphaq oligonucleotides 1 (-203/-175) and 2 (-174/-152) using HEL cell extracts demonstrated protein binding that was due to early growth response factor EGR-1 at two sites. Mutations in either EGR-1 site markedly decreased the gene activity, indicating functional relevance. Mutation of consensus E-Box motif (-185/-180) had no effect. Reduction in the expression of endogenous EGR-1 with antisense oligonucleotide to EGR-1 inhibited PMA-induced Galphaq transcription. Correspondingly, Egr-1 deficient mouse platelets also showed approximately 50% reduction in the Galphaq expression relative to wild-type platelets.

    Conclusions: These studies suggest that Galphaq gene is regulated during PMA-induced megakaryocytic differentiation by EGR-1, an early growth response transcription factor that regulates a wide array of genes and plays a major role in diverse activities, including cell proliferation, differentiation and apoptosis, and in vascular response to injury and atherosclerosis.

    Funded by: NHLBI NIH HHS: R01 HL56724

    Journal of thrombosis and haemostasis : JTH 2006;4;12;2678-86

  • ARF6 activation by Galpha q signaling: Galpha q forms molecular complexes with ARNO and ARF6.

    Giguère P, Rochdi MD, Laroche G, Dupré E, Whorton MR, Sunahara RK, Claing A, Dupuis G and Parent JL

    Service de Rhumatologie, Département de Médecine, Faculté de Médecine and Centre de Recherche Clinique, Université de Sherbrooke, 3001 12e Avenue Nord, Fleurimont, Quebec, Canada.

    G protein-coupled receptors (GPCRs) are widely expressed hepta-helical receptors with tightly regulated pleiotropic effects. ADP-Ribosylation Factor 6 (ARF6) plays an important role in GPCR trafficking and is the subject of intense research. However, the mechanisms underlying activation and regulation of ARF6 by GPCRs are poorly characterized. Here we report that Galpha(q) signaling leads to the activation of ARF6. Stimulation of the TPbeta receptor triggered ARF6 activation which was completely inhibited by the RGS domain of GRK2 known to specifically bind and sequester Galpha(q). Co-immunoprecipitation studies revealed that ARNO (a guanine nucleotide exchange factor for ARF6) and ARF6 formed complexes preferentially with activated Galpha(q) compared to non-activated Galpha(q). Formation of the Galpha(q) complexes with ARNO and ARF6 was detected early and was optimal after 30 min of receptor stimulation corresponding with the profile of ARF6 activation. Interestingly, binding experiments using purified proteins showed that Galpha(q) interacted directly with ARNO. Galpha(q)-dependent TPbeta receptor-mediated activation of ARF6 resulted in phosphoinositol-4,5-bisphosphate production which was potently inhibited by dominant negative mutants of ARNO and ARF6. Furthermore, our data show that the expression of ARNO and ARF6 promoted, whereas dominant negative mutants of these proteins inhibited the internalization of the TPbeta receptor. This further elucidates our previous data on the PLCbeta- and PKC-independent mechanism involved in Galpha(q)-mediated internalization of the TPbeta receptor. Taken altogether, our results support a novel model where activated Galpha(q) forms molecular complexes with ARNO and ARF6, possibly through a direct interaction with ARNO, leading to ARF6 activation.

    Cellular signalling 2006;18;11;1988-94

  • Reversible heart failure in G alpha(q) transgenic mice.

    Jiang YP, Ballou LM, Lu Z, Li Wan, Kelly DJ, Cohen IS and Lin RZ

    Department of Medicine, Stony Brook University, Stony Brook, New York 11794, USA.

    For many patients with cardiac insufficiency, the disease progresses inexorably to organ dilatation, pump failure, and death. Although there are examples of reversible heart failure in man, our understanding of how the myocardium repairs itself is limited. A well defined animal model of reversible heart failure would allow us to better investigate these restorative processes. Receptors that activate Galpha(q), a signal transduction molecule in the heterotrimeric G protein superfamily, are thought to play a key role in the development of heart failure. We demonstrated previously that mice expressing a recombinant Galpha(q) protein, the activity of which can be turned on or off at will in cardiac myocytes, develop a dilated cardiomyopathy with generalized edema and heart failure following activation of the protein (Fan, G., Jiang, Y.-P., Lu, Z., Martin, D. W., Kelly, D. J., Zuckerman, J. M., Ballou, L. M., Cohen, I. S., and Lin, R. Z. (2005) J. Biol. Chem. 280, 40337-40346). Here we report that the contractile dysfunction and pathological structural changes in the myocardium improved significantly after termination of the Galpha(q) signal, even in animals with overt heart failure. Abnormalities in two proteins that regulate Ca(2+) handling in myocytes, phospholamban and the voltage-dependent L-type Ca(2+) channel, were also reversed, as was the increased expression of genes that are associated with heart failure. These results indicate that the heart has a substantial reparative capacity if the molecular signals responsible for the myocardial dysfunction can be identified and blocked.

    Funded by: NHLBI NIH HHS: HL28958, HL67101; NIDDK NIH HHS: DK62722

    The Journal of biological chemistry 2006;281;40;29988-92

  • Bacterial superantigens bypass Lck-dependent T cell receptor signaling by activating a Galpha11-dependent, PLC-beta-mediated pathway.

    Bueno C, Lemke CD, Criado G, Baroja ML, Ferguson SS, Rahman AK, Tsoukas CD, McCormick JK and Madrenas J

    The FOCIS Centre for Clinical Immunology and Immunotherapeutics, London, Ontario N6A 5K8, Canada.

    The paradigm to explain antigen-dependent T cell receptor (TCR) signaling is based on the activation of the CD4 or CD8 coreceptor-associated kinase Lck. It is widely assumed that this paradigm is also applicable to signaling by bacterial superantigens. However, these bacterial toxins can activate human T cells lacking Lck, suggesting the existence of an additional pathway of TCR signaling. Here we showed that this alternative pathway operates in the absence of Lck-dependent tyrosine-phosphorylation events and was initiated by the TCR-dependent activation of raft-enriched heterotrimeric Galpha11 proteins. This event, in turn, activated a phospholipase C-beta and protein kinase C-mediated cascade that turned on the mitogen-activated protein kinases ERK-1 and ERK-2, triggered Ca(2+) influx, and translocated the transcription factors NF-AT and NF-kappaB to the nucleus, ultimately inducing the production of interleukin-2 in Lck-deficient T cells. The triggering of this alternative pathway by superantigens suggests that these toxins use a G protein-coupled receptor as a coreceptor on T cells.

    Funded by: NIAMS NIH HHS: AR048848

    Immunity 2006;25;1;67-78

  • Levels of G-protein alpha q/11 subunits and of phospholipase C-beta(1-4), -gamma, and -delta1 isoforms in postmortem human brain caudate and cortical membranes: potential functional implications.

    López de Jesús M, Zalduegui A, Ruiz de Azúa I, Callado LF, Meana JJ and Sallés J

    Department of Pharmacology, Faculty of Pharmacy, University of the Basque Country, Spain.

    The levels of expression of G-protein alpha(q/11) (Galpha(q/11)) subunits and PLC-beta(1-4), -gamma, and -delta(1) isoforms were quantified by Western blot analysis in order to establish their contribution to the patterns of PLC functioning reported here. Quantitative measurements of the levels of Galpha(q/11) subunits in each region were obtained by comparison with known amounts of Escherichia coli expressed recombinant Galpha(q) subunits. Quantitative analysis indicated that Galpha(q/11) subunits are abundant polypeptides in human brain, with values ranging from about 1200 ng/mg in cerebral cortex to close to 900 ng/mg of membrane protein in caudate. In cerebral cortical membranes, the PLC-beta(1) isoform was more abundant than in caudate membranes. The highest levels of PLC-beta(2) expression were detected in caudate membranes. PLC-beta(3) was little expressed, and there were no significant differences in the relative values between both brain regions. Finally, the levels of the PLC-beta(4) isoform were significantly lower in caudate than in cortical membranes. It is concluded that although most of these data represent relative, not absolute, measures of protein levels within these regions, they contribute nonetheless to the significant differences observed in signaling capacities through the PLC system in both human brain regions.

    Neurochemistry international 2006;49;1;72-9

  • The LIFEdb database in 2006.

    Mehrle A, Rosenfelder H, Schupp I, del Val C, Arlt D, Hahne F, Bechtel S, Simpson J, Hofmann O, Hide W, Glatting KH, Huber W, Pepperkok R, Poustka A and Wiemann S

    Division Molecular Genome Analysis, German Cancer Research Center, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany. a.mehrle@dkfz.de

    LIFEdb (http://www.LIFEdb.de) integrates data from large-scale functional genomics assays and manual cDNA annotation with bioinformatics gene expression and protein analysis. New features of LIFEdb include (i) an updated user interface with enhanced query capabilities, (ii) a configurable output table and the option to download search results in XML, (iii) the integration of data from cell-based screening assays addressing the influence of protein-overexpression on cell proliferation and (iv) the display of the relative expression ('Electronic Northern') of the genes under investigation using curated gene expression ontology information. LIFEdb enables researchers to systematically select and characterize genes and proteins of interest, and presents data and information via its user-friendly web-based interface.

    Nucleic acids research 2006;34;Database issue;D415-8

  • Loss of association between activated Galpha q and Gbetagamma disrupts receptor-dependent and receptor-independent signaling.

    Evanko DS, Thiyagarajan MM, Takida S and Wedegaertner PB

    Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, 233 S. 10th St., 839 BLSB, Philadelphia, PA 19107, USA.

    The G protein subunit, betagamma, plays an important role in targeting alpha subunits to the plasma membrane and is essential for binding and activation of the heterotrimer by heptahelical receptors. Mutation of residues in the N-terminal alpha-helix of alpha s and alpha q that contact betagamma in the crystal structure of alpha i reduces binding between alpha and betagamma, inhibits plasma membrane targeting and palmitoylation of the alpha subunit, and results in G proteins that fail to couple receptor activation to stimulation of effector. Overexpression of betagamma can recover this loss of signaling through Gs but not Gq. In fact, a single mutation (I25A) in alpha q can block alpha q-mediated generation of inositol phosphates. Function is not recovered by betagamma overexpression nor myristoylation directed plasma membrane localization. Introduction of a Q209L activating mutation with I25A results in a constitutively active alpha q as expected, but surprisingly a R183C activating mutation does not result in constitutive activity when present with I25A. Examination of binding between alpha and betagamma via a pull down assay shows that the N-terminal betagamma-binding mutations inhibit alpha-betagamma binding significantly more than the R183C or Q209L activating mutations do. Moreover, introduction of the I25A mutation into alpha q RC disrupts co-immunoprecipitation with PLCbeta1. Taken together, results presented here suggest that alpha-betagamma binding is necessary at a point downstream from receptor activation of the heterotrimeric G protein for signal transduction by alpha q.

    Funded by: NIGMS NIH HHS: GM56444, R01 GM056444-02, R01 GM056444-03, R01 GM056444-04, R01 GM056444-05, R01 GM056444-06, R01 GM056444-07

    Cellular signalling 2005;17;10;1218-28

  • Proteasome involvement in the degradation of the G(q) family of Galpha subunits.

    Johansson BB, Minsaas L and Aragay AM

    Department of Biomedicine, Faculty of Medicine, University of Bergen, Norway.

    Metabolically unstable proteins are involved in a multitude of regulatory networks, including those that control cell signaling, the cell cycle and in many responses to physiological stress. In the present study, we have determined the stability and characterized the degradation process of some members of the G(q) class of heterotrimeric G proteins. Pulse-chase experiments in HEK293 cells indicated a rapid turnover of endogenously expressed Galpha(q) and overexpressed Galpha(q) and Galpha(16) subunits. Pretreatment with proteasome inhibitors attenuated the degradation of both G alpha subunits. In contrast, pretreatment of cells with inhibitors of lysosomal proteases and nonproteasomal cysteine proteases had very little effect on the stability of the proteins. Significantly, the turnover of these proteins is not affected by transient activation of their associated receptors. Fractionation studies showed that the rates of Galpha(q) and Galpha16 degradation are accelerated in the cytosol. In fact, we show that a mutant Galpha(q) which lacks its palmitoyl modification site, and which is localized almost entirely in the cytoplasm, has a marked increase in the rate of degradation. Taken together, these results suggest that the G(q) class proteins are degraded through the proteasome pathway and that cellular localization and/or other protein interactions determine their stability.

    The FEBS journal 2005;272;20;5365-77

  • Protease-activated receptors in hemostasis, thrombosis and vascular biology.

    Coughlin SR

    Cardiovascular Research Institute, Department of Medicine, University of California, San Francisco, CA 94143-0130, USA. coughlin@cvrimail.ucsf.edu

    The coagulation cascade and protease-activated receptors (PARs) together provide an elegant mechanism that links mechanical information in the form of tissue injury to cellular responses. These receptors appear to largely account for the cellular effects of thrombin and can mediate signaling to other trypsin-like proteases. An important role for PARs in hemostasis and thrombosis is established in animal models, and studies in knockout mice and nonhuman primates raise the question of whether PAR inhibition might offer an appealing new approach to the prevention and treatment of thrombosis. PARs may also trigger inflammatory responses to tissue injury. For example, PAR activation on endothelial cells and perhaps sensory afferents can trigger local accumulation of leukocytes and platelets and transudation of plasma. However, panoply of signaling systems and cell types orchestrates inflammatory responses, and efforts to define the relative importance and roles of PARs in various inflammatory processes are just beginning. Lastly, roles for PARs in blood vessel formation and other processes during embryonic development are emerging, and whether these reflect new roles for the coagulation cascade and/or PAR signaling to other proteases remains to be explored.

    Journal of thrombosis and haemostasis : JTH 2005;3;8;1800-14

  • Galphaq potentiation of adenylate cyclase type 9 activity through a Ca2+/calmodulin-dependent pathway.

    Cumbay MG and Watts VJ

    Purdue University, Department of Medicinal Chemistry and Molecular Pharmacology, 575 Stadium Mall Drive, West Lafayette, IN 47907-2051, USA.

    Adenylate cyclase (EC type 9 (AC9) activity has been shown to be inhibited by PMA activation of novel protein kinase C (nPKC) isoforms. In the current study the effect on AC9 activity of activating PKC in physiological relevant manner was examined. Contrary to the anticipated inhibitory effect of activating PKCs through Gq-coupled receptors, activation of transiently expressed Gq-coupled serotonin 5-HT2A or muscarinic M5 receptors resulted in the potentiation of isoproterenol-stimulated cyclic AMP accumulation in HEK293 cells stably expressing AC9 (HEK-AC9). Consistent with Gq-mediated activation of PKC, the addition of the PKC inhibitor bisindolylmaleimide further potentiated isoproterenol-stimulated cyclic AMP accumulation. Expression of a constitutively active mutant of Galphaq in HEK-AC9 cells also produced an enhancement in basal and isoproterenol-stimulated cyclic AMP accumulation. We also examined the role of Galphaq-mediated release of intracellular Ca2+ on the observed potentiation of AC9 activity, by depleting intracellular Ca2+ stores with thapsigargin. In Ca2+-depleted HEK-AC9 cells, activation of transiently expressed M5 receptors resulted in inhibition of isoproterenol-stimulated cyclic AMP accumulation that was blocked by bisindolylmaleimide, indicating that M5 potentiation of AC9 activity requires Ca2+. This prompted us to examine the effects of the calmodulin antagonist W7 and the Ca2+/calmodulin-dependent kinase II (CaMK II) inhibitor KN-93. Pretreating cells with W7 and KN-93 significantly inhibited M5-mediated potentiation of isoproterenol-stimulated cyclic AMP accumulation in HEK-AC9 cells, suggesting that Galphaq potentiation of AC9 activity involves Ca2+/calmodulin and CaMK II. This data provides evidence for Ca2+-mediated potentiation of AC9 activity.

    Funded by: NIGMS NIH HHS: T32GM008737; NIMH NIH HHS: MH60397

    Biochemical pharmacology 2005;69;8;1247-56

  • Functional consequences of G alpha 13 mutations that disrupt interaction with p115RhoGEF.

    Grabocka E and Wedegaertner PB

    Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, USA.

    The G-protein alpha subunit, alpha(13), regulates cell growth and differentiation through the monomeric Rho GTPase. Alpha(13) activates Rho through direct stimulation of the guanine nucleotide exchange factor p115RhoGEF, which contains a regulator of G-protein signaling homology domain (RH) in its N-terminus. Through its RH domain, p115RhoGEF also functions as a GAP for G alpha(13). The mechanism for the G alpha(13)/p115RhoGEF interaction is not well understood. Here, we determined specific alpha(13) residues important for its interaction with p115RhoGEF. GST-pulldowns and co-immunoprecipitation assays revealed that individually mutating alpha(13) residues Lys204, Glu229, or Arg232 to opposite charge residues disrupts the interaction of activated alpha(13) with the RH domain of p115RhoGEF or full-length p115RhoGEF. We further demonstrate that mutation of Glu229, and to a lesser extent Lys204 or Arg232, disrupts the ability of activated alpha(13) to induce the recruitment of p115RhoGEF to the plasma membrane (PM) and to activate Rho-mediated serum response element-luciferase gene transcription. Interestingly, an alpha(13) mutant where a conserved Gly was mutated to a Ser (G205S) retained its ability to bind to p115RhoGEF, induce p115RhoGEF recruitment to the PM, and activate Rho-dependent signaling, even though identical Gly to Ser mutations in other alpha disrupt their interaction with regulator of G-protein signaling (RGS) proteins. These results demonstrate that, whereas several features of a typical alpha/RGS interaction are preserved in the alpha(13)/p115RhoGEF interaction, there are also significant differences.

    Funded by: NIGMS NIH HHS: GM62884, R01 GM062884

    Oncogene 2005;24;13;2155-65

  • Agonist-induced tyrosine phosphorylation of Gq/G11 alpha requires the intact structure of membrane domains.

    Matousek P, Durchánková D, Svandová I, Novotny J and Svoboda P

    Institute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic.

    Stimulation of receptors coupled to G(q)/G(11) protein may induce phosphorylation on a tyrosine residue of the alpha subunit of this G protein, which is an essential event for G(q)/G(11) activation. Here we observed that in HEK293 cells stably expressing high levels of thyrotropin-releasing hormone (TRH) receptors and G(11)alpha protein the maximal tyrosine phosphorylation of G(q)/G(11)alpha was reached within 10 min of TRH stimulation and then it faded away at longer time periods of agonist exposure. The G(q)/G(11)alpha protein levels did not change during this treatment. Incubation of intact cells with beta-cyclodextrin (beta CD) for 40 min prior to hormone exposure significantly decreased the rapid transient tyrosine phosphorylation. Subsequent replenishment of cholesterol levels reversed the former negative effect of beta CD. Isolation of caveolin-enriched, detergent-resistant membrane domains indicated destruction of these structures in beta CD-treated cells. These data indicate that the preserved integrity of plasma membrane domains/caveolae is required for complete agonist-induced phosphorylation of G(q)/G(11)alpha.

    Biochemical and biophysical research communications 2005;328;2;526-32

  • beta-Arrestin 1 and Galphaq/11 coordinately activate RhoA and stress fiber formation following receptor stimulation.

    Barnes WG, Reiter E, Violin JD, Ren XR, Milligan G and Lefkowitz RJ

    Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.

    beta-Arrestins were initially shown, in conjunction with G protein-coupled receptor kinases, to be involved in the desensitization and internalization of activated seven-transmembrane receptors. Recently, beta-arrestin 2 has been shown to act as a signal mediator in mitogen-activated protein kinase cascades and to play a positive regulatory role in chemotaxis. We now show that beta-arrestin 1 is required to activate the small GTPase RhoA leading to the re-organization of stress fibers following the activation of the angiotensin II type 1A receptor. This angiotensin II type 1A receptor-directed RhoA activation and stress fiber formation also require the activation of the heterotrimeric G protein G(alphaq/11). Whereas neither beta-arrestin 1 nor G(alphaq/11) activation alone is sufficient to robustly activate RhoA, the concurrent recruitment of beta-arrestin 1 and activation of G(alphaq/11) leads to full activation of RhoA and to the subsequent formation of stress fibers.

    Funded by: NHLBI NIH HHS: HL 16037, HL 70631

    The Journal of biological chemistry 2005;280;9;8041-50

  • G alpha(q)-coupled receptors in human atrium function through protein kinase C epsilon and delta.

    Kilts JD, Grocott HP and Kwatra MM

    Department of Anesthesiology, Duke University Medical Center, Durham, NC 27710, USA.

    Cardiac G alpha(q)-coupled receptors (such as endothelin, angiotensin, and alpha1-adrenergic receptors) mediate cardiac inotropy and chronotropy, as well as the development of hypertrophy. These receptors signal through protein kinase C (PKC), a family of 12 isozymes including PKC alpha, beta I, beta II, gamma, delta, epsilon, theta, eta, lambda, iota, zeta, and mu. Of these PKC isozymes, alpha, beta II, gamma, epsilon, delta, and zeta have been implicated in signaling through cardiac G alpha(q)-coupled receptors in various animal models. However, the profile of which isozymes are activated by a given G alpha(q)-coupled receptor varies among animal species. Thus, these results can not be extrapolated to human heart. In this study, we examine PKC isozymes activated by three different G alpha(q)-coupled receptors in human atrial tissue. Live atrial appendages obtained from the operating room were sliced and treated with agonists of G alpha(q)-coupled receptors, and cellular redistribution of PKC isozymes was examined by immunoblotting. We find that stimulation of G alpha(q)-coupled receptors in human atrium activates PKC epsilon and delta only, under both acute (5 min) and longer (35 min) stimulations. Further, PKC epsilon and delta exhibit distinct subcellular redistribution patterns; while both translocate to the plasma membrane upon G alpha(q) stimulation, PKC delta also redistributes to mitochondria. We conclude that PKC epsilon and delta are the main PKC isozymes involved in G alpha(q)-mediated signaling in human atria.

    Funded by: NIA NIH HHS: AG00029, AG15817

    Journal of molecular and cellular cardiology 2005;38;2;267-76

  • Polycystin-1 activates the calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway.

    Puri S, Magenheimer BS, Maser RL, Ryan EM, Zien CA, Walker DD, Wallace DP, Hempson SJ and Calvet JP

    Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA.

    Regulation of intracellular Ca(2+) mobilization has been associated with the functions of polycystin-1 (PC1) and polycystin-2 (PC2), the protein products of the PKD1 and PKD2 genes. We have now demonstrated that PC1 can activate the calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway through Galpha(q) -mediated activation of phospholipase C (PLC). Transient transfection of HEK293T cells with an NFAT promoter-luciferase reporter demonstrated that membrane-targeted PC1 constructs containing the membrane proximal region of the C-terminal tail, which includes the heterotrimeric G protein binding and activation domain, can stimulate NFAT luciferase activity. Inhibition of glycogen synthase kinase-3beta by LiCl treatment further increased PC1-mediated NFAT activity. PC1-mediated activation of NFAT was completely inhibited by the calcineurin inhibitor, cyclosporin A. Cotransfection of a construct expressing the Galpha(q) subunit augmented PC1-mediated NFAT activity, whereas the inhibitors of PLC (U73122) and the inositol trisphosphate and ryanodine receptors (xestospongin and 2-aminophenylborate) and a nonspecific Ca(2+) channel blocker (gadolinium) diminished PC1-mediated NFAT activity. PC2 was not able to activate NFAT. An NFAT-green fluorescent protein nuclear localization assay demonstrated that PC1 constructs containing the C-tail only or the entire 11-transmembrane spanning region plus C-tail induced NFAT-green fluorescent protein nuclear translocation. NFAT expression was demonstrated in the M-1 mouse cortical collecting duct cell line and in embryonic and adult mouse kidneys by reverse transcriptase-PCR and immunolocalization. These data suggest a model in which PC1 signaling leads to a sustained add elevation of intracellular Ca(2+) mediated by PC1 activation of Galpha(q) followed by PLC activation, release of Ca(2+) from intracellular stores, and activation of store-operated Ca(2+) entry, thus activating calcineurin and NFAT.

    Funded by: NIDDK NIH HHS: DK 53763, DK 57301

    The Journal of biological chemistry 2004;279;53;55455-64

  • Characterization of the GRK2 binding site of Galphaq.

    Day PW, Tesmer JJ, Sterne-Marr R, Freeman LC, Benovic JL and Wedegaertner PB

    Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, 233 S. 10th St., Philadelphia, PA 19107, USA.

    Heterotrimeric guanine nucleotide-binding proteins (G proteins) transmit signals from membrane bound G protein-coupled receptors (GPCRs) to intracellular effector proteins. The G(q) subfamily of Galpha subunits couples GPCR activation to the enzymatic activity of phospholipase C-beta (PLC-beta). Regulators of G protein signaling (RGS) proteins bind to activated Galpha subunits, including Galpha(q), and regulate Galpha signaling by acting as GTPase activating proteins (GAPs), increasing the rate of the intrinsic GTPase activity, or by acting as effector antagonists for Galpha subunits. GPCR kinases (GRKs) phosphorylate agonist-bound receptors in the first step of receptor desensitization. The amino termini of all GRKs contain an RGS homology (RH) domain, and binding of the GRK2 RH domain to Galpha(q) attenuates PLC-beta activity. The RH domain of GRK2 interacts with Galpha(q/11) through a novel Galpha binding surface termed the "C" site. Here, molecular modeling of the Galpha(q).GRK2 complex and site-directed mutagenesis of Galpha(q) were used to identify residues in Galpha(q) that interact with GRK2. The model identifies Pro(185) in Switch I of Galpha(q) as being at the crux of the interface, and mutation of this residue to lysine disrupts Galpha(q) binding to the GRK2-RH domain. Switch III also appears to play a role in GRK2 binding because the mutations Galpha(q)-V240A, Galpha(q)-D243A, both residues within Switch III, and Galpha(q)-Q152A, a residue that structurally supports Switch III, are defective in binding GRK2. Furthermore, GRK2-mediated inhibition of Galpha(q)-Q152A-R183C-stimulated inositol phosphate release is reduced in comparison to Galpha(q)-R183C. Interestingly, the model also predicts that residues in the helical domain of Galpha(q) interact with GRK2. In fact, the mutants Galpha(q)-K77A, Galpha(q)-L78D, Galpha(q)-Q81A, and Galpha(q)-R92A have reduced binding to the GRK2-RH domain. Finally, although the mutant Galpha(q)-T187K has greatly reduced binding to RGS2 and RGS4, it has little to no effect on binding to GRK2. Thus the RH domain A and C sites for Galpha(q) interaction rely on contacts with distinct regions and different Switch I residues in Galpha(q).

    Funded by: NIGMS NIH HHS: GM44944, GM62884, R01 GM062884

    The Journal of biological chemistry 2004;279;51;53643-52

  • Single-cell imaging of intracellular Ca2+ and phospholipase C activity reveals that RGS 2, 3, and 4 differentially regulate signaling via the Galphaq/11-linked muscarinic M3 receptor.

    Tovey SC and Willars GB

    Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, United Kingdom.

    Using single cell, real-time imaging, this study compared the impact of members of the B/R4 subfamily of the regulators of G-protein signaling (RGS) (RGS2, -3, and -4) on receptor-mediated inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], diacylglycerol, and Ca2+ signaling. In human embryonic kidney (HEK) 293 cells expressing recombinant Galpha(q/11)-coupled muscarinic M3 receptors, transient coexpression of RGS proteins with fluorescently-tagged biosensors for either Ins(1,4,5)P3 or diacylglycerol demonstrated that RGS2 and 3 inhibited receptor-mediated events. Although gross indices of signaling were unaffected by RGS4, it slowed the rate of increase in Ins(1,4,5)P3 levels. At equivalent levels of expression, myc-tagged RGS proteins showed inhibitory activity on the order RGS3 > or = RGS2 > RGS4. In HEK293 cells, stable expression of myc-tagged RGS2, -3, or -4 at equivalent levels also inhibited phosphoinositide and Ca2+ signaling by endogenously expressed muscarinic M3 receptors in the order RGS3 > or = RGS2 > RGS4. In these cells, RGS2 or -3 reduced receptor-mediated inositol phosphate generation in cell populations and reduced both the magnitude and kinetics (rise-time) of single cell Ca2+ signals. Furthermore, at low levels of receptor activation, oscillatory Ca2+ signals were dampened or abolished, whereas at higher levels, RGS2 and -3 promoted the conversion of more stable Ca2+ elevations into oscillatory signals. Despite little or no effect on responses to maximal receptor activation, RGS4 produced effects on the magnitude, kinetics, and oscillatory behavior of Ca2+ signaling at submaximal levels that were consistent with those of RGS2 and -3.

    Molecular pharmacology 2004;66;6;1453-64

  • From ORFeome to biology: a functional genomics pipeline.

    Wiemann S, Arlt D, Huber W, Wellenreuther R, Schleeger S, Mehrle A, Bechtel S, Sauermann M, Korf U, Pepperkok R, Sültmann H and Poustka A

    Molecular Genome Analysis, German Cancer Research Center, 69120 Heidelberg, Germany. s.wiemann@dkfz.de

    As several model genomes have been sequenced, the elucidation of protein function is the next challenge toward the understanding of biological processes in health and disease. We have generated a human ORFeome resource and established a functional genomics and proteomics analysis pipeline to address the major topics in the post-genome-sequencing era: the identification of human genes and splice forms, and the determination of protein localization, activity, and interaction. Combined with the understanding of when and where gene products are expressed in normal and diseased conditions, we create information that is essential for understanding the interplay of genes and proteins in the complex biological network. We have implemented bioinformatics tools and databases that are suitable to store, analyze, and integrate the different types of data from high-throughput experiments and to include further annotation that is based on external information. All information is presented in a Web database (http://www.dkfz.de/LIFEdb). It is exploited for the identification of disease-relevant genes and proteins for diagnosis and therapy.

    Genome research 2004;14;10B;2136-44

  • Effects of G-protein mutations on skin color.

    Van Raamsdonk CD, Fitch KR, Fuchs H, de Angelis MH and Barsh GS

    Department of Genetics, Stanford University School of Medicine, Stanford, California 94305-5323, USA.

    A new class of dominant dark skin (Dsk) mutations discovered in a screen of approximately 30,000 mice is caused by increased dermal melanin. We identified three of four such mutations as hypermorphic alleles of Gnaq and Gna11, which encode widely expressed Galphaq subunits, act in an additive and quantitative manner, and require Ednrb. Interactions between Gq and Kit receptor tyrosine kinase signaling can mediate coordinate or independent control of skin and hair color. Our results provide a mechanism that can explain several aspects of human pigmentary variation and show how polymorphism of essential proteins and signaling pathways can affect a single physiologic system.

    Nature genetics 2004;36;9;961-8

  • CCL3, acting via the chemokine receptor CCR5, leads to independent activation of Janus kinase 2 (JAK2) and Gi proteins.

    Mueller A and Strange PG

    School of Animal and Microbial Sciences, University of Reading, Whiteknights, Reading RG6 6AJ, UK.

    The interaction of the chemokine receptor, CCR5, expressed in recombinant cells, with different G proteins was investigated and CCR5 was found to interact with Gi, Go and Gq species. Interaction with Gi leads to G protein activation, whereas Gq does not seem to be activated. Additionally, CCR5 activation also leads to phosphorylation of Janus kinase 2 (JAK2). Activation of JAK2 is independent of Gi or Gq activation. Gi protein activation was not prevented by inhibition of JAK, showing that heterotrimeric G protein activation and activation of the JAK/signal transducer and activator of transcription (STAT) pathway are independent of each other.

    FEBS letters 2004;570;1-3;126-32

  • Gbetagamma signaling and Ca2+ mobilization co-operate synergistically in a Sos and Rac-dependent manner in the activation of JNK by Gq-coupled receptors.

    Chan AS and Wong YH

    Department of Biochemistry, the Biotechnology Research Institute, and the Molecular Neuroscience Center, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.

    The mechanism by which G(q)-coupled receptors stimulate the c-Jun N-terminal kinase (JNK) activity has not been fully delineated. Here, we showed that stimulation of endogenous G(q)-coupled receptors in human hepatocarcinoma HepG2 cells resulted in an Src family kinase- and Ca(2+)-dependent JNK activation. Cos-7 cells transfected with HA-tagged JNK and various G(q)-coupled receptors also exhibited similar characteristics and provided further evidence for the involvement of Gbetagamma, an upstream intermediate for Src family kinases. The Ca(2+) and Gbetagamma signals operate in a high degree of independence. Transient expression of Gbetagamma subunits and elevation of cytoplasmic Ca(2+) level by thapsigargin activated JNK in a synergistic fashion. JNK activities triggered by G(q)-coupled receptors, Gbetagamma and thapsigargin were all suppressed by dominant negative (DN) mutants of Son of sevenless (Sos) and Rac. We propose that the co-operative effect between Gbetagamma-mediated signaling and the increased intracellular Ca(2+) level represents a robust mechanism for the stimulation of JNK by G(q)-coupled receptors.

    Cellular signalling 2004;16;7;823-36

  • Activation of platelet-activating factor receptor-coupled G alpha q leads to stimulation of Src and focal adhesion kinase via two separate pathways in human umbilical vein endothelial cells.

    Deo DD, Bazan NG and Hunt JD

    Department of Biochemistry and Molecular Biology, Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA.

    Platelet-activating factor (PAF), a phospholipid second messenger, has diverse physiological functions, including responses in differentiated endothelial cells to external stimuli. We used human umbilical vein endothelial cells (HUVECs) as a model system. We show that PAF activated pertussis toxin-insensitive G alpha(q) protein upon binding to its seven transmembrane receptor. Elevated cAMP levels were observed via activation of adenylate cyclase, which activated protein kinase A (PKA) and was attenuated by a PAF receptor antagonist, blocking downstream activity. Phosphorylation of Src by PAF required G alpha(q) protein and adenylate cyclase activation; there was an absolute requirement of PKA for PAF-induced Src phosphorylation. Immediate (1 min) PAF-induced STAT-3 phosphorylation required the activation of G alpha(q) protein, adenylate cyclase, and PKA, and was independent of these intermediates at delayed (30 min) and prolonged (60 min) PAF exposure. PAF activated PLC beta 3 through its G alpha(q) protein-coupled receptor, whereas activation of phospholipase C gamma 1 (PLC gamma 1) by PAF was independent of G proteins but required the involvement of Src at prolonged PAF exposure (60 min). We demonstrate for the first time in vascular endothelial cells: (i) the involvement of signaling intermediates in the PAF-PAF receptor system in the induction of TIMP2 and MT1-MMP expression, resulting in the coordinated proteolytic activation of MMP2, and (ii) a receptor-mediated signal transduction cascade for the tyrosine phosphorylation of FAK by PAF. PAF exposure induced binding of p130(Cas), Src, SHC, and paxillin to FAK. Clearly, PAF-mediated signaling in differentiated endothelial cells is critical to endothelial cell functions, including cell migration and proteolytic activation of MMP2.

    Funded by: NIEHS NIH HHS: ES00358-03

    The Journal of biological chemistry 2004;279;5;3497-508

  • Phospholipase C-independent activation of glycogen synthase kinase-3beta and C-terminal Src kinase by Galphaq.

    Fan, Ballou LM and Lin RZ

    Department of Medicine, Stony Brook University, Stony Brook, New York 11794, USA.

    It is generally thought that activation of phospholipase Cbeta (PLCbeta) by Galphaq accounts for most of the effects of Gq-coupled receptors. Here we describe a novel effect of Galphaq that is independent of the PLCbeta pathway. Expression of the constitutively active Galphaq mutant Galphaq(Q209L) promoted an increase in glycogen synthase kinase-3beta (GSK-3beta) activity that was associated with increased phosphorylation of Tyr216 on GSK-3beta. Galphaq(Q209L)-AA, a mutant that cannot activate PLCbeta, also induced GSK-3beta activation and phosphorylation of Tyr216. We speculate that the protein-tyrosine kinase Csk (C-terminal Src kinase), which is also activated by Galphaq(Q209L) and Galphaq(Q209L)-AA, acts upstream of GSK-3beta. Expression of Csk accentuated the activation of GSK-3beta by Galphaq(Q209L), whereas catalytically inactive Csk blocked GSK-3beta activation by Galphaq(Q209L). Recombinant Csk phosphorylated and activated GSK-3beta in vitro, and GSK-3beta coprecipitated with Csk from cell lysates. These results suggest that activation of Csk and GSK-3beta by Galphaq may contribute to the physiological and pathological effects of Gq-coupled receptors.

    Funded by: NIDDK NIH HHS: R01 DK62722

    The Journal of biological chemistry 2003;278;52;52432-6

  • RGS16 inhibits signalling through the G alpha 13-Rho axis.

    Johnson EN, Seasholtz TM, Waheed AA, Kreutz B, Suzuki N, Kozasa T, Jones TL, Brown JH and Druey KM

    Molecular Signal Transduction Section, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases/National Institute of Health, Rockville, MD 20852, USA.

    G alpha 13 stimulates the guanine nucleotide exchange factors (GEFs) for Rho, such as p115Rho-GEF. Activated Rho induces numerous cellular responses, including actin polymerization, serum response element (SRE)-dependent gene transcription and transformation. p115Rho-GEF contains a Regulator of G protein Signalling domain (RGS box) that confers GTPase activating protein (GAP) activity towards G alpha 12 and G alpha 13 (ref. 3). In contrast, classical RGS proteins (such as RGS16 and RGS4) exhibit RGS domain-dependent GAP activity on G alpha i and G alpha q, but not G alpha 12 or G alpha 13 (ref 4). Here, we show that RGS16 inhibits G alpha 13-mediated, RhoA-dependent reversal of stellation and SRE activation. The RGS16 amino terminus binds G alpha 13 directly, resulting in translocation of G alpha 13 to detergent-resistant membranes (DRMs) and reduced p115Rho-GEF binding. RGS4 does not bind G alpha 13 or attenuate G alpha 13-dependent responses, and neither RGS16 nor RGS4 affects G alpha 12-mediated signalling. These results elucidate a new mechanism whereby a classical RGS protein regulates G alpha 13-mediated signal transduction independently of the RGS box.

    Funded by: NIGMS NIH HHS: GM36927

    Nature cell biology 2003;5;12;1095-103

  • G beta gamma mediates the interplay between tubulin dimers and microtubules in the modulation of Gq signaling.

    Popova JS and Rasenick MM

    Department of Physiology and Biophysics, College of Medicine, University of Illinois, Chicago, Illinois 60612-7342, USA.

    Agonist stimulation causes tubulin association with the plasma membrane and activation of PLC beta 1 through direct interaction with, and transactivation of, G alpha q. Here we demonstrate that G beta gamma interaction with tubulin down-regulates this signaling pathway. Purified G beta gamma, alone or with phosphatidylinositol 4,5-bisphosphate (PIP2), inhibited carbachol-evoked membrane recruitment of tubulin and G alpha q transactivation by tubulin. Polymerization of microtubules elicited by G beta gamma overrode tubulin translocation to the membrane in response to carbachol stimulation. G beta gamma sequestration of tubulin reduced the inhibition of PLC beta 1 observed at high tubulin concentration. G beta 1 gamma 2 interacted preferentially with tubulin-GDP, whereas G alpha q was transactivated by tubulin-GTP. Prenylation of the gamma 2 polypeptide was required for G beta gamma/tubulin interaction. Both confocal microscopy and coimmunoprecipitation studies revealed the spatiotemporal pattern of G beta gamma/tubulin interaction during carbachol stimulation of neuroblastoma SK-N-SH cells. In resting cells G beta gamma localized predominantly at the cell membrane, whereas tubulin was found in well defined microtubules in the cytosol. Within 2 min of agonist exposure, a subset of tubulin translocated to the plasma membrane and colocalized with G beta. Fifteen min post-carbachol addition, tubulin and G beta colocalized in vesicle-like structures in the cytosol. G beta/tubulin colocalization increased after pretreatment of cells with the microtubule-depolymerizing agent, colchicine, and was inhibited by taxol. Taxol also inhibited carbachol-induced PIP2 hydrolysis. It is suggested that G beta gamma/tubulin interaction mediates internalization of membrane-associated tubulin at the offset of PLC beta 1 signaling. Newly cytosolic G beta gamma/tubulin complexes might promote microtubule polymerization attenuating further tubulin association with the plasma membrane. Thus G protein-coupled receptors might evoke G alpha and G beta gamma to orchestrate regulation of phospholipase signaling by tubulin dimers and control of cell shape by microtubules.

    Funded by: NIA NIH HHS: AG 15482; NIMH NIH HHS: MH 39595

    The Journal of biological chemistry 2003;278;36;34299-308

  • Differential interaction of GRK2 with members of the G alpha q family.

    Day PW, Carman CV, Sterne-Marr R, Benovic JL and Wedegaertner PB

    Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, 233 South 10th Street, Philadelphia, Pennsylvania 19107, USA.

    Regulators of G protein signaling (RGS) proteins bind to active G alpha subunits and accelerate the rate of GTP hydrolysis and/or block interaction with effector molecules, thereby decreasing signal duration and strength. RGS proteins are defined by the presence of a conserved 120-residue region termed the RGS domain. Recently, it was shown that the G protein-coupled receptor kinase 2 (GRK2) contains an RGS domain that binds to the active form of G alpha(q). Here, the ability of GRK2 to interact with other members of the G alpha(q) family, G alpha(11), G alpha(14), and G alpha(16), was tested. The signaling of all members of the G alpha(q) family, with the exception of G alpha(16), was inhibited by GRK2. Immunoprecipitation of full-length GRK2 or pull down of GST-GRK2-(45-178) resulted in the detection of G alpha(q), but not G alpha(16), in an activation-dependent manner. Moreover, activated G alpha(16) failed to promote plasma membrane (PM) recruitment of a GRK2-(45-178)-GFP fusion protein. Assays with chimeric G alpha(q)(-)(16) subunits indicated that the C-terminus of G alpha(q) mediates binding to GRK2. Despite showing no interaction with GRK2, G alpha(16) does interact with RGS2, in both inositol phosphate and PM recruitment assays. Thus, GRK2 is the first identified RGS protein that discriminates between members of the G alpha(q) family, while another RGS protein, RGS2, binds to both G alpha(q) and G alpha(16).

    Funded by: NCI NIH HHS: T32-CA09662; NIGMS NIH HHS: GM44944, GM47417, GM628884

    Biochemistry 2003;42;30;9176-84

  • Functional characterization of human receptors for short chain fatty acids and their role in polymorphonuclear cell activation.

    Le Poul E, Loison C, Struyf S, Springael JY, Lannoy V, Decobecq ME, Brezillon S, Dupriez V, Vassart G, Van Damme J, Parmentier M and Detheux M

    Euroscreen, rue Adrienne Bolland 47, 6041 Gosselies, Laboratory of Molecular Immunology, Rega Institute for Medical Research, Catholic University of Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium.

    Short chain fatty acids (SCFAs), including acetate, propionate, and butyrate, are produced at high concentration by bacteria in the gut and subsequently released in the bloodstream. Basal acetate concentrations in the blood (about 100 microm) can further increase to millimolar concentrations following alcohol intake. It was known previously that SCFAs can activate leukocytes, particularly neutrophils. In the present work, we have identified two previously orphan G protein-coupled receptors, GPR41 and GPR43, as receptors for SCFAs. Propionate was the most potent agonist for both GPR41 and GPR43. Acetate was more selective for GPR43, whereas butyrate and isobutyrate were more active on GPR41. The two receptors were coupled to inositol 1,4,5-trisphosphate formation, intracellular Ca2+ release, ERK1/2 activation, and inhibition of cAMP accumulation. They exhibited, however, a differential coupling to G proteins; GPR41 coupled exclusively though the Pertussis toxin-sensitive Gi/o family, whereas GPR43 displayed a dual coupling through Gi/o and Pertussis toxin-insensitive Gq protein families. The broad expression profile of GPR41 in a number of tissues does not allow us to infer clear hypotheses regarding its biological functions. In contrast, the highly selective expression of GPR43 in leukocytes, particularly polymorphonuclear cells, suggests a role in the recruitment of these cell populations toward sites of bacterial infection. The pharmacology of GPR43 matches indeed the effects of SCFAs on neutrophils, in terms of intracellular Ca2+ release and chemotaxis. Such a neutrophil-specific SCFA receptor is potentially involved in the development of a variety of diseases characterized by either excessive or inefficient neutrophil recruitment and activation, such as inflammatory bowel diseases or alcoholism-associated immune depression. GPR43 might therefore constitute a target allowing us to modulate immune responses in these pathological situations.

    The Journal of biological chemistry 2003;278;28;25481-9

  • Activated G alpha q inhibits p110 alpha phosphatidylinositol 3-kinase and Akt.

    Ballou LM, Lin HY, Fan G, Jiang YP and Lin RZ

    Research Service, Department of Veterans Affairs Medical Center, Northport, New York 11768, USA.

    Some Gq-coupled receptors have been shown to antagonize growth factor activation of phosphatidylinositol 3-kinase (PI3K) and its downstream effector, Akt. We used a constitutively active Galphaq(Q209L) mutant to explore the effects of Galphaq activation on signaling through the PI3K/Akt pathway. Transient expression of Galphaq(Q209L) in Rat-1 fibroblasts inhibited Akt activation induced by platelet-derived growth factor or insulin treatment. Expression of Galphaq(Q209L) also attenuated Akt activation promoted by coexpression of constitutively active PI3K in human embryonic kidney 293 cells. Galphaq(Q209L) had no effect on the activity of an Akt mutant in which the two regulatory phosphorylation sites were changed to acidic amino acids. Inducible expression of Galphaq(Q209L) in a stably transfected 293 cell line caused a decrease in PI3K activity in p110alpha (but not p110beta) immunoprecipitates. Receptor activation of Galphaq also selectively inhibited PI3K activity in p110alpha immunoprecipitates. Active Galphaq still inhibited PI3K/Akt in cells pretreated with the phospholipase C inhibitor U73122. Finally, Galphaq(Q209L) co-immunoprecipitated with the p110alpha-p85alpha PI3K heterodimer from lysates of COS-7 cells expressing these proteins, and incubation of immunoprecipitated Galphaq(Q209L) with purified recombinant p110alpha-p85alpha in vitro led to a decrease in PI3K activity. These results suggest that agonist binding to Gq-coupled receptors blocks Akt activation via the release of active Galphaq subunits that inhibit PI3K. The inhibitory mechanism seems to be independent of phospholipase C activation and might involve an inhibitory interaction between Galphaq and p110alpha PI3K.

    Funded by: NIDDK NIH HHS: R01 DK62722

    The Journal of biological chemistry 2003;278;26;23472-9

  • G beta 5.RGS7 inhibits G alpha q-mediated signaling via a direct protein-protein interaction.

    Witherow DS, Tovey SC, Wang Q, Willars GB and Slepak VZ

    Department of Molecular and Cellular Pharmacology and the Neuroscience Program, University of Miami School of Medicine, Miami, Florida 33136, USA.

    A subfamily of regulators of G protein signaling (RGS) proteins consisting of RGS6, -7, -9, and -11 is characterized by the presence of a unique Ggamma-like domain through which they form obligatory dimers with the G protein subunit Gbeta5 in vivo. In Caenorhabditis elegans, orthologs of Gbeta5.RGS dimers are implicated in regulating both Galphai and Galphaq signaling, and in cell-based assays these dimers regulate Galphai/o- and Galphaq/11-mediated pathways. However, initial studies with purified Gbeta5.RGS6 or Gbeta5.RGS7 showed that they only serve as GTPase activating proteins for Galphao. Pull-down assays and co-immunoprecipitation with these dimers failed to detect their binding to either Galphao or Galphaq, indicating that the interaction might require additional factors present in vivo. Here, we asked if the RGS7.Gbeta5 complex binds to Galphaq using fluorescence resonance energy transfer (FRET) in transiently transfected mammalian cells. RGS7, Gbeta5, and Galpha subunits were tagged with yellow variants of green fluorescent protein. First we confirmed the functional activity of the fusion proteins by co-immunoprecipitation and also their effect on signaling. Second, we again demonstrate the interaction between RGS7 and Gbeta5 using FRET. Finally, using both FRET spectroscopy on cell suspensions and microscopy of individual cells, we showed FRET between the yellow fluorescence protein-tagged RGS7.Gbeta5 complex and cyan fluorescence protein-tagged Galphaq, indicating a direct interaction between these molecules.

    Funded by: NEI NIH HHS: EY 12982; NIGMS NIH HHS: R01 GM 600019, R01 GM060019, R01 GM060019-01, R01 GM060019-02, R01 GM060019-02S1, R01 GM060019-03, R01 GM060019-03S1

    The Journal of biological chemistry 2003;278;23;21307-13

  • Heterotrimeric G alpha q/G alpha 11 proteins function upstream of vascular endothelial growth factor (VEGF) receptor-2 (KDR) phosphorylation in vascular permeability factor/VEGF signaling.

    Zeng H, Zhao D, Yang S, Datta K and Mukhopadhyay D

    Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA.

    Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) functions by activating two receptor-tyrosine kinases, Flt-1 (VEGF receptor (VEGFR)-1) and KDR (VEGFR-2), both of which are selectively expressed on primary vascular endothelium. KDR is responsible for VPF/VEGF-stimulated endothelial cell proliferation and migration, whereas Flt-1 down-modulates KDR-mediated endothelial cell proliferation. Our most recent works show that pertussis toxin-sensitive G proteins and Gbetagamma subunits are required for Flt-1-mediated down-regulation of human umbilical vein endothelial cell (HUVEC) proliferation and that Gq/11 proteins are required for KDR-mediated RhoA activation and HUVEC migration. In this study, we demonstrate that Gq/11 proteins are also required for VPF/VEGF-stimulated HUVEC proliferation. Our results further indicate that Gq/11 proteins specifically mediate KDR signaling such as intracellular Ca2+ mobilization rather than Flt-1-induced CDC42 activation and that a Gq/11 antisense oligonucleotide completely inhibits MAPK phosphorylation induced by KDR but has no effect on Flt-1-induced MAPK activation. More importantly, we demonstrate that Gq/11 proteins interact with KDR in vivo, and the interaction of Gq/11 proteins with KDR does not require KDR tyrosine phosphorylation. Surprisingly, the Gq/11 antisense oligonucleotide completely inhibits VPF/VEGF-stimulated KDR phosphorylation. Expression of a constitutively active mutant of G11 but not Gq can cause phosphorylation of KDR and MAPK. In addition, a Gbetagamma minigene, hbetaARK1(495), inhibits VPF/VEGF-stimulated HUVEC proliferation, MAPK phosphorylation, and intracellular Ca2+ mobilization but has no effect on KDR phosphorylation. Taken together, this study demonstrates that Gq/11 proteins mediate KDR tyrosine phosphorylation and KDR-mediated HUVEC proliferation through interaction with KDR.

    Funded by: NCI NIH HHS: CA78383; NHLBI NIH HHS: HL072178, HL70567

    The Journal of biological chemistry 2003;278;23;20738-45

  • Rapid activation of Ras by fluid flow is mediated by Galpha(q) and Gbetagamma subunits of heterotrimeric G proteins in human endothelial cells.

    Gudi S, Huvar I, White CR, McKnight NL, Dusserre N, Boss GR and Frangos JA

    Department of Bioengineering, University of California at San Diego, La Jolla, USA.

    Objective: Temporal gradients in fluid shear stress have been shown to induce a proatherogenic phenotype in endothelial cells. The biomechanical mechanism(s) that enables the endothelium to respond to fluid shear stress requires rapid activation and signal transduction. The small G protein Ras has been identified as an early link between rapid mechanotransduction events and the effects of shear stress on downstream signal-transduction cascades. The aim of this study was to elucidate the upstream mechanotransduction signaling events mediating the rapid activation of Ras by fluid shear stress in human endothelial cells.

    Direct measurement of Ras-bound GTP and GDP showed that fluid-flow activation of Ras was rapid (10-fold within 5 seconds) and dose dependent on shear stress magnitude. Treatment with protein tyrosine kinase inhibitors or pertussis toxin did not significantly affect flow-induced Ras activation. However, activation was inhibited by transient transfection with antisense to Galpha(q) or the Gbetagamma scavenger beta-adrenergic receptor kinase carboxy terminus. Transfection with several Gbetagamma subunit isoforms revealed flow-induced Ras activation was most effectively enhanced by Gbeta1gamma2.

    Conclusions: These results suggest that the rapid, shear-induced activation of Ras is mediated by Galpha(q) through the activity of Gbetagamma subunits in human vascular endothelial cells.

    Funded by: NHLBI NIH HHS: HL-40696

    Arteriosclerosis, thrombosis, and vascular biology 2003;23;6;994-1000

  • Identification of tetratricopeptide repeat 1 as an adaptor protein that interacts with heterotrimeric G proteins and the small GTPase Ras.

    Marty C, Browning DD and Ye RD

    Department of Pharmacology, College of Medicine, University of Illinois, Chicago, Illinois 60612, USA.

    The biological functions of heterotrimeric G proteins and small GTPases are modulated by both extracellular stimuli and intracellular regulatory proteins. Using Saccharomyces cerevisiae two-hybrid screening, we identified tetratricopeptide repeat 1 (TPR1), a 292-amino-acid protein with three TPR motifs, as a Galpha16-binding protein. The interaction was confirmed both in vitro and in transfected mammalian cells, where TPR1 also binds to several other Galpha proteins. TPR1 was found to interact with Ha-Ras preferentially in its active form. Overexpression of TPR1 promotes accumulation of active Ras. TPR1 was found to compete with the Ras-binding domain (RBD) of Raf-1 for binding to the active Ras, suggesting that it may also compete with Ras GTPase-activating protein, thus contributing to the accumulation of GTP-bound Ras. Expression of Galpha16 strongly enhances the interaction between TPR1 and Ras. Removal of the TPR1 N-terminal 112 residues abolishes potentiation by Galpha16 while maintaining the interaction with Galpha16 and the ability to discriminate active Ras from wild-type Ras. We have also observed that LGN, a Galphai-interacting protein with seven TPR motifs, binds Ha-Ras. Thus, TPR1 is a novel adaptor protein for Ras and selected Galpha proteins that may be involved in protein-protein interaction relating to G-protein signaling.

    Funded by: NIAID NIH HHS: AI33503, AI40176, R01 AI033503, R01 AI040176, R56 AI033503, R56 AI040176

    Molecular and cellular biology 2003;23;11;3847-58

  • Galphaq-coupled receptor internalization specifically induced by Galphaq signaling. Regulation by EBP50.

    Rochdi MD and Parent JL

    Service de Rhumatologie, Faculté de Médecine and Centre de Recherche Clinique, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.

    In the present report, we investigated the effect of ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) expression on the agonist-induced internalization of the thromboxane A(2) beta receptor (TPbeta receptor). Interestingly, we found that EBP50 almost completely blocked TPbeta receptor internalization, which could not be reversed by overexpression of G protein-coupled receptor (GPCR) kinases and arrestins. Because we recently demonstrated that EBP50 can bind to and inhibit Galpha(q), we next studied whether Galpha(q) signaling could induce TPbeta receptor internalization, addressing the long standing question about the relationship between GPCR signaling and their internalization. Expression of a constitutively active Galpha(q) mutant (Galpha(q)-R183C) resulted in a robust internalization of the TPbeta receptor, which was unaffected by expression of dominant negative mutants of arrestin-2 and -3, but inhibited by expression of EBP50 or dynamin-K44A, a dominant negative mutant of dynamin. Phospholipase Cbeta and protein kinase C did not appear to significantly contribute to internalization of the TPbeta receptor, suggesting that Galpha(q) induces receptor internalization through a phospholipase Cbeta- and protein kinase C-independent pathway. Surprisingly, there appears to be specificity in Galpha protein-mediated GPCR internalization. Galpha(q)-R183C also induced the internalization of CXCR4 (Galpha(q)-coupled), whereas it failed to do so for the beta(2)-adrenergic receptor (Galpha(s)-coupled). Moreover, Galpha(s)-R201C, a constitutively active form of Galpha(s), had no effect on internalization of the TPbeta, CXCR4, and beta(2)-adrenergic receptors. Thus, we showed that Galpha protein signaling can lead to internalization of GPCRs, with specificity in both the Galpha proteins and GPCRs that are involved. Furthermore, a new function has been described for EBP50 in its capacity to inhibit receptor endocytosis.

    The Journal of biological chemistry 2003;278;20;17827-37

  • Human brain synembryn interacts with Gsalpha and Gqalpha and is translocated to the plasma membrane in response to isoproterenol and carbachol.

    Klattenhoff C, Montecino M, Soto X, Guzmán L, Romo X, García MA, Mellstrom B, Naranjo JR, Hinrichs MV and Olate J

    Departamento de Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Concepción, Chile.

    Heterotrimeric G-proteins transduce signals from heptahelical transmembrane receptors to different effector systems, regulating diverse complex intracellular pathways and functions. In brain, facilitation of depolarization-induced neurotransmitter release for synaptic transmission is mediated by Gsalpha and Gqalpha. To identify effectors for Galpha-proteins, we performed a yeast two-hybrid screening of a human brain cDNA library, using the human Galphas protein as a bait. We identified a protein member of the synembryn family as one of the interacting proteins. Extending the study to other Galpha subunits, we found that Gqalpha also interacts with synembryn, and these interactions were confirmed by in vitro pull down studies and by in vivo confocal laser microscopy analysis. Furthermore, synembryn was shown to translocate to the plasma membrane in response to carbachol and isoproterenol. This study supports recent findings in C. elegans where, through genetic studies, synembryn was shown to act together with Gqalpha regulating neuronal transmitter release. Based on these observations, we propose that synembryn is playing a similar role in human neuronal cells.

    Journal of cellular physiology 2003;195;2;151-7

  • 5-HT2A and 5-HT2C receptors and their atypical regulation properties.

    Van Oekelen D, Luyten WH and Leysen JE

    Johnson and Johnson Pharmaceutical, p/a Janssen Pharmaceutica, Turnhoutseweg 30, B-2340 Beerse, Belgium.

    The 5-HT(2A) and 5-HT(2C) receptors belong to the G-protein-coupled receptor (GPCR) superfamily. GPCRs transduce extracellular signals to the interior of cells through their interaction with G-proteins. The 5-HT(2A) and 5-HT(2C) receptors mediate effects of a large variety of compounds affecting depression, schizophrenia, anxiety, hallucinations, dysthymia, sleep patterns, feeding behaviour and neuro-endocrine functions. Binding of such compounds to either 5-HT(2) receptor subtype induces processes that regulate receptor sensitivity. In contrast to most other receptors, chronic blockade of 5-HT(2A) and 5-HT(2C) receptors leads not to an up- but to a (paradoxical) down-regulation. This review deals with published data involving such non-classical regulation of 5-HT(2A) and 5-HT(2C) receptors obtained from in vivo and in vitro studies. The underlying regulatory processes of the agonist-induced regulation of 5-HT(2A) and 5-HT(2C) receptors, commonly thought to be desensitisation and resensitisation, are discussed. The atypical down-regulation of both 5-HT(2) receptor subtypes by antidepressants, antipsychotics and 5-HT(2) antagonists is reviewed. The possible mechanisms of this paradoxical down-regulation are discussed, and a new hypothesis on possible heterologous regulation of 5-HT(2A) receptors is proposed.

    Life sciences 2003;72;22;2429-49

  • The orphan G protein-coupled receptor GPR40 is activated by medium and long chain fatty acids.

    Briscoe CP, Tadayyon M, Andrews JL, Benson WG, Chambers JK, Eilert MM, Ellis C, Elshourbagy NA, Goetz AS, Minnick DT, Murdock PR, Sauls HR, Shabon U, Spinage LD, Strum JC, Szekeres PG, Tan KB, Way JM, Ignar DM, Wilson S and Muir AI

    Department of Metabolic Diseases, GlaxoSmithKline, Research Triangle Park, North Carolina 27709, USA. cpb15172@gsk.com

    GPR40 is a member of a subfamily of homologous G protein-coupled receptors that include GPR41 and GPR43 and that have no current function or ligand ascribed. Ligand fishing experiments in HEK293 cells expressing human GPR40 revealed that a range of saturated and unsaturated carboxylic acids with carbon chain lengths greater than six were able to induce an elevation of [Ca(2+)](i), measured using a fluorometric imaging plate reader. 5,8,11-Eicosatriynoic acid was the most potent fatty acid tested, with a pEC(50) of 5.7. G protein coupling of GPR40 was examined in Chinese hamster ovary cells expressing the G alpha(q/i)-responsive Gal4-Elk1 reporter system. Expression of human GPR40 led to a constitutive induction of luciferase activity, which was further increased by exposure of the cells to eicosatriynoic acid. Neither the constitutive nor ligand-mediated luciferase induction was inhibited by pertussis toxin treatment, suggesting that GPR40 was coupled to G alpha(q/11.) Expression analysis by quantitative reverse transcription-PCR showed that GPR40 was specifically expressed in brain and pancreas, with expression in rodent pancreas being localized to insulin-producing beta-cells. These data suggest that some of the physiological effects of fatty acids in pancreatic islets and brain may be mediated through a cell-surface receptor.

    The Journal of biological chemistry 2003;278;13;11303-11

  • Free fatty acids regulate insulin secretion from pancreatic beta cells through GPR40.

    Itoh Y, Kawamata Y, Harada M, Kobayashi M, Fujii R, Fukusumi S, Ogi K, Hosoya M, Tanaka Y, Uejima H, Tanaka H, Maruyama M, Satoh R, Okubo S, Kizawa H, Komatsu H, Matsumura F, Noguchi Y, Shinohara T, Hinuma S, Fujisawa Y and Fujino M

    Discovery Research Laboratories I, Pharmaceutical Research Division, Takeda Chemical Industries, Ltd, Wadai 10, Tsukuba, Ibaraki 300-4293, Japan.

    Diabetes, a disease in which carbohydrate and lipid metabolism are regulated improperly by insulin, is a serious worldwide health issue. Insulin is secreted from pancreatic beta cells in response to elevated plasma glucose, with various factors modifying its secretion. Free fatty acids (FFAs) provide an important energy source as nutrients, and they also act as signalling molecules in various cellular processes, including insulin secretion. Although FFAs are thought to promote insulin secretion in an acute phase, this mechanism is not clearly understood. Here we show that a G-protein-coupled receptor, GPR40, which is abundantly expressed in the pancreas, functions as a receptor for long-chain FFAs. Furthermore, we show that long-chain FFAs amplify glucose-stimulated insulin secretion from pancreatic beta cells by activating GPR40. Our results indicate that GPR40 agonists and/or antagonists show potential for the development of new anti-diabetic drugs.

    Nature 2003;422;6928;173-6

  • Mammalian Ric-8A (synembryn) is a heterotrimeric Galpha protein guanine nucleotide exchange factor.

    Tall GG, Krumins AM and Gilman AG

    Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75390-9041, USA.

    The activation of heterotrimeric G proteins is accomplished primarily by the guanine nucleotide exchange activity of ligand-bound G protein-coupled receptors. The existence of nonreceptor guanine nucleotide exchange factors for G proteins has also been postulated. Yeast two-hybrid screens with Galpha(o) and Galpha(s) as baits were performed to identify binding partners of these proteins. Two mammalian homologs of the Caenorhabditis elegans protein Ric-8 were identified in these screens: Ric-8A (Ric-8/synembryn) and Ric-8B. Purification and biochemical characterization of recombinant Ric-8A revealed that it is a potent guanine nucleotide exchange factor for a subset of Galpha proteins including Galpha(q), Galpha(i1), and Galpha(o), but not Galpha(s). The mechanism of Ric-8A-mediated guanine nucleotide exchange was elucidated. Ric-8A interacts with GDP-bound Galpha proteins, stimulates release of GDP, and forms a stable nucleotide-free transition state complex with the Galpha protein; this complex dissociates upon binding of GTP to Galpha.

    Funded by: NIGMS NIH HHS: GM34497

    The Journal of biological chemistry 2003;278;10;8356-62

  • Regulation of GTP-binding protein alpha q (Galpha q) signaling by the ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50).

    Rochdi MD, Watier V, La Madeleine C, Nakata H, Kozasa T and Parent JL

    Service de Rhumatologie, Faculté de Médecine and Centre de Recherche Clinique-CHUS, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.

    Although ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a PDZ domain-containing protein known to bind to various channels, receptors, cytoskeletal elements, and cytoplasmic proteins, there is still very little evidence for a role of EBP50 in the regulation of receptor signal transduction. In this report, we show that EBP50 inhibits the phospholipase C (PLC)-beta-mediated inositol phosphate production of a Galpha(q)-coupled receptor as well as PLC-beta activation by the constitutively active Galpha(q)-R183C mutant. Coimmunoprecipitation experiments revealed that EBP50 interacts with Galpha(q) and to a greater extent with Galpha(q)-R183C. Agonist stimulation of the thromboxane A(2) receptor (TP receptor) resulted in an increased interaction between EBP50 and Galpha(q), suggesting that EBP50 preferentially interacts with activated Galpha(q). We also demonstrate that EBP50 inhibits Galpha(q) signaling by preventing the interaction between Galpha(q) and the TP receptor and between activated Galpha(q) and PLC-beta1. Investigation of the EBP50 regions involved in Galpha(q) binding indicated that its two PDZ domains are responsible for this interaction. This study constitutes the first demonstration of an interaction between a G protein alpha subunit and another protein through a PDZ domain, with broad implications in the regulation of diverse physiological systems.

    The Journal of biological chemistry 2002;277;43;40751-9

  • Negative and positive regulatory epitopes in the C-terminal domains of the human B1 and B2 bradykinin receptor subtypes determine receptor coupling efficacy to G(q/11)-mediated [correction of G(9/11)-mediated] phospholipase Cbeta activity.

    Kang DS and Leeb-Lundberg LM

    Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas 78229-3900, USA.

    The human B1 bradykinin (BK) receptor (B1R) is more efficacious than the human B2 BK receptor (B2R) in both ligand-independent and agonist-dependent coupling to G(q/11)-mediated phospholipase Cbeta activity. In fact, B1R is constitutively active, whereas B2R exhibits little if any constitutive activity. To evaluate the role of the C-terminal domain in receptor G(q/11) coupling, we constructed chimeric and C-terminally truncated receptors. The slopes of the increase in basal and agonist-dependent cellular phosphoinositide hydrolysis as a function of receptor density in transiently transfected human embryonic kidney 293 cells provided parameters of receptor coupling. Exchanging the C-terminal domains between the two receptors revealed that these domains are largely responsible for the difference in coupling. B1R truncation showed that this receptor does not directly depend on the C-terminal domain for efficient coupling, although coupling is dramatically augmented by residues in the membrane-distal portion of the domain downstream from Tyr(327). On the other hand, coupling of B2R is absolutely dependent on a membrane-proximal epitope in the C-terminal domain upstream from Lys(315). This epitope is adjacent to a basic residue, Arg(311), which exerts an inhibitory effect on coupling. Arg(311) is not conserved in B1R, and complementary mutations in B2R and B1R showed that this residue, together with previously identified serines and threonines, acts to attenuate the coupling efficacy of B2R. Therefore, the C-terminal domain participates intimately in the efficacy of B1R and B2R G(q/11) coupling by contributing both positive and negative regulatory epitopes.

    Funded by: NIGMS NIH HHS: GM41659

    Molecular pharmacology 2002;62;2;281-8

  • Potent activation of RhoA by Galpha q and Gq-coupled receptors.

    Chikumi H, Vázquez-Prado J, Servitja JM, Miyazaki H and Gutkind JS

    Oral and Pharyngeal Cancer Branch, NIDCR/National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892-4330, USA.

    Heterotrimeric G proteins of the G(i), G(s), and G(q) family control a wide array of physiological functions primarily by regulating the activity of key intracellular second messenger-generating systems. alpha subunits of the G(12) family, Galpha(12) and Galpha(13), however, can promote cellular responses that are independent of conventional second messengers but that result from the activation of small GTP-binding proteins of the Rho family and their downstream targets. These findings led to the identification of a novel family of guanine-nucleotide exchange factors (GEFs) that provides a direct link between Galpha(12/13) and Rho stimulation. Recent observations suggest that many cellular responses elicited by Galpha(q) and its coupled receptors also require the functional activity of Rho. However, available evidence suggests that Galpha(q) may act on pathways downstream from Rho rather than by promoting Rho activation. These seemingly conflicting observations and the recent development of sensitive assays to assess the in vivo levels of active Rho prompted us to ask whether Galpha(q) and its coupled receptors can stimulate endogenous Rho. Here we show that the expression of activated forms of Galpha(q) and the stimulation of G(q)-coupled receptors or chimeric Galpha(q) molecules that respond to G(i)-linked receptors can promote a robust activation of endogenous Rho in HEK-293T cells. Interestingly, this response was not prevented by molecules interfering with the ability of Galpha(13) to stimulate its linked RhoGEFs, together suggesting the existence of a novel molecular mechanism by which Galpha(q) and the large family of G(q)-coupled receptors can regulate the activity of Rho and its downstream signaling pathways.

    The Journal of biological chemistry 2002;277;30;27130-4

  • Cloning and characterization of a novel regulator of G protein signalling in human platelets.

    Gagnon AW, Murray DL and Leadley RJ

    Cardiovascular Drug Discovery, Aventis Pharmaceuticals, 500 Arcola Road, Collegeville, PA 19426, USA.

    In an effort to understand the modulation of G protein-coupled receptor (GPCR)-mediated signalling in platelets, we sought to identify which regulators of G protein signalling proteins (RGSs) are present in human platelets. Using degenerate oligonucleotides, we performed RT-PCR with human platelet and megakaryocytic cell line RNA. In addition to confirming the presence of several known RGS transcripts, we found a novel RGS domain-containing transcript in platelet RNA. Northern blot analysis of multiple human tissues indicates that this transcript is most abundantly expressed in platelets compared to other tissues examined. Full-length cloning of this novel RGS, which we now term RGS18, demonstrates that this transcript is predicted to encode a 235-amino acid protein that is most closely related to RGS5 (46% identity) and that has approximately 30-40% identity to other RGS proteins. RGS18 is expressed in platelet, leukocyte, and megakaryocyte cell lines and binds to endogenous Galphai1, Galphai2, Galphai3, and Galphaq but not Galphaz, Galphas or Galpha12 in vitro.

    Cellular signalling 2002;14;7;595-606

  • Functional characterization of the G protein regulator RGS13.

    Johnson EN and Druey KM

    Molecular Signal Transduction Section, Laboratory of Allergic Diseases, NIAID, National Institutes of Health, Rockville, Maryland 20852, USA.

    The signaling cascades evoked by G protein-coupled receptors are a predominant mechanism of cellular communication. The regulators of G protein signaling (RGS) comprise a family of proteins that attenuate G protein-mediated signal transduction. Here we report the characterization of RGS13, the smallest member of the RGS family, which has been cloned from human lung. RGS13 has been found most abundantly in human tonsil, followed by thymus, lung, lymph node, and spleen. RGS13 is a GTPase-activating protein for Galpha(i) and Galpha(o) but not Galpha(s). RGS13 binds Galpha(q) in the presence of aluminum magnesium fluoride, suggesting that it bears GTPase-activating protein activity toward Galpha(q). RGS13 blocks MAPK activity induced by Galpha(i)- or Galpha(q)-coupled receptors. RGS13 also attenuates GTPase-deficient Galpha(q) (Galpha(q)QL) mediated cAMP response element activation but not transcription evoked by constitutively active Galpha(12) or Galpha(13). Surprisingly, RGS13 inhibits cAMP generation elicited by stimulation of the beta(2)-adrenergic receptor. These data suggest that RGS13 may regulate Galpha(i)-, Galpha(q)-, and Galpha(s)-coupled signaling cascades.

    The Journal of biological chemistry 2002;277;19;16768-74

  • A small sequence in the third intracellular loop of the VPAC(1) receptor is responsible for its efficient coupling to the calcium effector.

    Langer I, Vertongen P, Perret J, Waelbroeck M and Robberecht P

    Department of Biological Chemistry and Nutrition, Faculty of Medicine, Université Libre de Bruxelles, B-1070 Brussels, Belgium. ilanger@ulb.ac.be

    The stimulatory effect of VIP on intracellular calcium concentration ([Ca(2+)](i)) has been investigated in Chinese hamster ovary cells stably transfected with the reporter gene aequorin, and expressing human VPAC(1), VPAC(2), chimeric VPAC(1)/VPAC(2), or mutated receptors. The VIP-induced [Ca(2+)](i) increase was linearly correlated with receptor density and was higher in cells expressing VPAC(1) receptors than in cells expressing a similar VPAC(2) receptor density. The study was performed to establish the receptor sequence responsible for that difference. VPAC(1)/VPAC(2) chimeric receptors were first used for a broad positioning: those having the third intracellular loop (IC(3)) of the VPAC(1) or of the VPAC(2) receptor behaved, in that respect, phenotypically like VPAC(1) and VPAC(2) receptor, respectively. Replacement in the VPAC(2) receptor of the sequence 315-318 (VGGN) within the IC(3) by its VPAC(1) receptor counterpart 328-331 (IRKS) and the introduction of VGGN in state of IRKS in VPAC(1) was sufficient to mimic the VPAC(1) and VPAC(2) receptor characteristics, respectively. Thus, a small sequence in the IC(3) of the VPAC(1) receptor, probably through interaction with G(alphai) and G(alphaq) proteins, is responsible for the efficient agonist-stimulated [Ca(2+)](i) increase.

    Molecular endocrinology (Baltimore, Md.) 2002;16;5;1089-96

  • Identification of G-proteins coupling to the vasoactive intestinal peptide receptor VPAC(1) using immunoaffinity chromatography: evidence for precoupling.

    Martin Shreeve S

    Department of Pharmacology, University of Vermont, Burlington 05405, USA. Stephen.Shreeve@uvm.edu

    VPAC(1) receptor subtype-specific G-protein interactions were identified using a strategy that exploits an essential initial signaling event, namely the functional and physical association of the receptor with G-protein. An immunoaffinity purification column was constructed using a previously characterized antibody that had been raised against the first extracellular loop of the VPAC(1) receptor. VPAC(1)/G-protein complexes were solubilized from membranes and copurified. Receptor and Galpha-proteins were detected in eluates using (125)I-VIP labeling and immunoblotting, respectively. Human VPAC(1) transfected in HEK293 cells couples to Gs but not Gi3, Gi1/2, or Gq. Rat VPAC(1) in brain membranes is coupled to Gs and Gi3. Rat VPAC(1) in lung membranes couples to Gs, Gi3, and Gq. Pretreatment of membranes with VIP increased the level of all G-pro c1f teins copurifying with VPAC(1). Immunoaffinity chromatography also revealed VPAC(1) receptor precoupling to G-protein in the absence of VIP pretreatment. This was confirmed using a cross-linking procedure to capture VIP receptor/G-protein complexes in the native membrane milieu prior to solubilization. Precoupling suggests that there is a significant basal level of VPAC(1) receptor activity especially in cells, such as some human malignant tumor cells, that express high levels of receptor.

    Biochemical and biophysical research communications 2002;290;4;1300-7

  • A unique fold of phospholipase C-beta mediates dimerization and interaction with G alpha q.

    Singer AU, Waldo GL, Harden TK and Sondek J

    Department of Pharmacology, The University of North Carolina at Chapel Hill, North Carolina 27599, USA.

    GTP-bound subunits of the Gq family of G alpha subunits directly activate phospholipase C-beta (PLC-beta) isozymes to produce the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. PLC-betas are GTPase activating proteins (GAPs) that also promote the formation of GDP-bound, inactive G beta subunits. Both phospholipase activation by G alpha-GTP subunits and GAP activity require a C-terminal region unique to PLC-beta isozymes. The crystal structure of the C-terminal region from an avian PLC-beta, determined at 2.4 A resolution, reveals a novel fold composed almost entirely of three long helices forming a coiled-coil that dimerizes along its long axis in an antiparallel orientation. The dimer interface is extensive ( approximately 3,200 A(2)), and, based on gel exclusion chromatography, full length PLC-betas are dimeric, indicating that PLC-betas likely function as dimers. Sequence conservation, mutational data and molecular modeling show that an electrostatically positive surface of the dimer contains the major determinants for binding G beta q. Effector dimerization, as highlighted by PLC-betas, provides a viable mechanism for regulating signaling cascades linked to heterotrimeric G proteins.

    Nature structural biology 2002;9;1;32-6

  • Human platelet Galphaq deficiency is associated with decreased Galphaq gene expression in platelets but not neutrophils.

    Gabbeta J, Vaidyula VR, Dhanasekaran DN and Rao AK

    Sol Sherry Thrombosis Research Center and Fels Center for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA 19140, USA.

    G-proteins play an important role in platelet signal transduction and regulate responses upon activation of G-protein coupled receptors (GPCR). We have previously reported a patient with impaired platelet responses associated with deficiency in platelet Galphaq. To understand the molecular basis for this defect, the cDNA sequence encoding Galphaq (1080 bp) was obtained by reverse-transcription and polymerase chain reaction of platelet RNA; the cDNA sequence showed no mutations in the patient. Platelet Galphaq mRNA levels were decreased by >50% compared to normal subjects; platelet Galphai2 mRNA levels were normal. Neutrophil calcium mobilization and elastase secretion, upon activation with several agonists, and neutrophil Galphaq mRNA and protein levels were normal. These studies demonstrate that the patient has a defect in Galphaq gene expression in platelets but not neutrophils, possibly due to defects in transcriptional regulation or mRNA stability, and suggest a hematopoietic-lineage specific defect.

    Funded by: NHLBI NIH HHS: R01HL 56724

    Thrombosis and haemostasis 2002;87;1;129-33

  • Multiplicity of mechanisms of serotonin receptor signal transduction.

    Raymond JR, Mukhin YV, Gelasco A, Turner J, Collinsworth G, Gettys TW, Grewal JS and Garnovskaya MN

    The Research Service of the Ralph H. Johnson Veterans Affairs Medical Center, Charleston, SC 29401, USA. raymondj@musc.edu

    The serotonin (5-hydroxytryptamine, 5-HT) receptors have been divided into 7 subfamilies by convention, 6 of which include 13 different genes for G-protein-coupled receptors. Those subfamilies have been characterized by overlapping pharmacological properties, amino acid sequences, gene organization, and second messenger coupling pathways. Post-genomic modifications, such as alternative mRNA splicing or mRNA editing, creates at least 20 more G-protein-coupled 5-HT receptors, such that there are at least 30 distinct 5-HT receptors that signal through G-proteins. This review will focus on what is known about the signaling linkages of the G-protein-linked 5-HT receptors, and will highlight some fascinating new insights into 5-HT receptor signaling.

    Funded by: NIDDK NIH HHS: DK02694, DK053981, DK52448, DK54720

    Pharmacology & therapeutics 2001;92;2-3;179-212

  • Biogenesis of G-protein mediated calcium signaling in human megakaryocytes.

    den Dekker E, Gorter G, van der Vuurst H, Heemskerk JW and Akkerman JW

    Department of Haematology, University Medical Center Utrecht, The Netherlands. e.dendekker@lab.azu.nl

    To understand how platelet signal transduction pathways develop during megakaryocytopoiesis, we isolated human stem cells from umbilical cord blood and cultured the cells in the presence of thrombopoietin (TPO). Based on the early expression of CD61 and late expression of CD42b, immature (CD61+/CD42b(low)) and mature (CD61+/ CD42b(high)) megakaryocytes were immunomagnetically purified and, together with stem cells (CD34+), characterized for Galpha-protein expression and agonist-induced [Ca2+]i increases. Megakaryocytopoiesis was accompanied by down-regulation of the 43 kDa and 46 kDa variants of G16alpha, constant expression of Gsalpha, and up-regulation of Gqalpha and Gialpha1/2. The increase in Gqalpha and Gialpha1/2 expression was accompanied by an increase in Ca2+ signaling triggered by thrombin and other agonists known to signal to Ca2+ via these G-proteins in platelets. The prostacyclin analog iloprost and TPO also induced [Ca2+]i increases, and the iloprost-induced Ca2+ response disappeared during maturation. These data reveal sharp changes in Ca2+ regulation during megakaryocytopoiesis.

    Thrombosis and haemostasis 2001;86;4;1106-13

  • Protein kinase A-mediated phosphorylation of serine 357 of the mouse prostacyclin receptor regulates its coupling to G(s)-, to G(i)-, and to G(q)-coupled effector signaling.

    Lawler OA, Miggin SM and Kinsella BT

    Department of Biochemistry, Conway Institute of Biomolecular and Biomedical Research, Merville House, University College Dublin, Belfield, Dublin 4, Ireland.

    The prostacyclin receptor (IP) is primarily coupled to G alpha(s)-dependent activation of adenylyl cyclase; however, a number of studies indicate that the IP may couple to other secondary effector systems perhaps in a species-specific manner. In the current study, we investigated the specificity of G protein:effector coupling by the mouse (m) IP overexpressed in human embryonic kidney 293 cells and endogenously expressed in murine erythroleukemia cells. The mIP exhibited efficient G alpha(s) coupling and concentration-dependent increases in cAMP generation in response to the IP agonist cicaprost; however, mIP also coupled to G alpha(i) decreasing the levels of cAMP in forskolin-treated cells. mIP coupling to G alpha(i) was pertussis toxin-sensitive and was dependent on protein kinase (PK) A activation status. In addition, the mIP coupled to phospholipase C (PLC) activation in a pertussis toxin-insensitive, G alpha(i)-, G beta gamma-, and PKC-independent but in a G alpha(q)- and PKA-dependent manner. Whole cell phosphorylation assays demonstrated that the mIP undergoes cicaprost-induced PKA phosphorylation. mIP(S357A), a site-directed mutant of mIP, efficiently coupled to G alpha(s) but failed to couple to G alpha(i) or to efficiently couple to G alpha(q):PLC. Moreover, mIP(S357A) did not undergo cicaprost-induced phosphorylation confirming that Ser(357) is the target residue for PKA-dependent phosphorylation. Finally, co-precipitation experiments permitted the detection of G alpha(s), G alpha(i), and G alpha(q) in the immunoprecipitates of mIP, whereas only G alpha(s) was co-precipitated with mIP(S357A) indicating that Ser(357) of mIP is essential for G alpha(i) and G alpha(q) interaction. Moreover, inhibition of PKA blocked co-precipitation of mIP with G alpha(i) or G alpha(q). Taken together our data indicate that the mIP, in addition to coupling to G alpha(s), couples to G alpha(i) and G alpha(q); however, G alpha(i) and G alpha(q) coupling is dependent on initial cicaprost-induced mIP:G alpha(s) coupling and phosphorylation of mIP by cAMP-dependent PKA where Ser(357) was identified as the target residue for PKA phosphorylation.

    The Journal of biological chemistry 2001;276;36;33596-607

  • Analysis of Gq protein alpha subunit mRNA expression in human monocytes: relevance of the purification step.

    Calò L, Milani M, Pagnin E, Davis P, Piccin A, Costa R, Plebani M and Semplicini A

    Department of Clinical and Experimental Medicine, Clinica Medica 4, University of Padova-Azienda Ospedaliera, Via Giustiniani, 2, 35128, Padua, Italy. renzcalo@unipd.it

    Background: Galphaq is a member of the Gq family of G proteins, which by stimulating the phospholipase Cbeta (PLCbeta)-IP(3)-Ca(++) mediated intracellular signaling systems controls some of the most fundamental cardiovascular cellular processes. The study of Galphaq is complicated by the presence of a pseudogene in human DNA, with signficant homology to the mRNA encoding the alpha subunit of Gq protein. The presence of this pseudogene will cause problems when the analysis of the Galphaq gene expression is based solely on RT-PCR. In this study, we report a simple method for avoiding unwanted amplification of the Galphaq pseudogene and the use of human monocytes as a readily available source for examining Galphaq.

    Methods: RT-PCR was carried out on RNA extracted from peripheral blood monocytes of 10 normal subjects using specific primers for Galphaq.

    Results: When several subjects' Galphaq was examined, the authentic Galphaq mRNA amplification product levels, as a ratio to unpurified pseudogene containing amplification products, declined by up to approximately 70%.

    Conclusion: Given the importance of Gq protein in cardiovascular signal transduction, it is fundamental to provide a reliable assessment of G alpha q gene expression. In addition to accurately assessing Galphaq levels, the use of circulating human monocytes as a useful source of Galphaq for investigating mechanisms involved in the regulation of vascular tone is shown.

    Clinica chimica acta; international journal of clinical chemistry 2001;309;1;13-8

  • G-protein signaling through tubby proteins.

    Santagata S, Boggon TJ, Baird CL, Gomez CA, Zhao J, Shan WS, Myszka DG and Shapiro L

    Ruttenberg Cancer Center, Structural Biology Program, Department of Physiology and Biophysics, Mount Sinai School of Medicine of New York University, 1425 Madison Avenue New York, NY 10029, USA.

    Dysfunction of the tubby protein results in maturity-onset obesity in mice. Tubby has been implicated as a transcription regulator, but details of the molecular mechanism underlying its function remain unclear. Here we show that tubby functions in signal transduction from heterotrimeric GTP-binding protein (G protein)-coupled receptors. Tubby localizes to the plasma membrane by binding phosphatidylinositol 4,5-bisphosphate through its carboxyl terminal "tubby domain." X-ray crystallography reveals the atomic-level basis of this interaction and implicates tubby domains as phosphorylated-phosphatidyl- inositol binding factors. Receptor-mediated activation of G protein alphaq (Galphaq) releases tubby from the plasma membrane through the action of phospholipase C-beta, triggering translocation of tubby to the cell nucleus. The localization of tubby-like protein 3 (TULP3) is similarly regulated. These data suggest that tubby proteins function as membrane-bound transcription regulators that translocate to the nucleus in response to phosphoinositide hydrolysis, providing a direct link between G-protein signaling and the regulation of gene expression.

    Science (New York, N.Y.) 2001;292;5524;2041-50

  • Physical and functional interactions of Galphaq with Rho and its exchange factors.

    Sagi SA, Seasholtz TM, Kobiashvili M, Wilson BA, Toksoz D and Brown JH

    Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0636, USA.

    Recent reports have shown that several heterotrimeric protein-coupled receptors that signal through Galpha(q) can induce Rho-dependent responses, but the pathways that mediate the interaction between Galpha(q) and Rho have not yet been identified. In this report we present evidence that Galpha(q) expressed in COS-7 cells coprecipitates with the Rho guanine nucleotide exchange factor (GEF) Lbc. Furthermore, Galpha(q) expression enhances Rho-dependent responses. Coexpressed Galpha(q) and Lbc have a synergistic effect on the Rho-dependent rounding of 1321N1 astrocytoma cells. In addition, serum response factor-dependent gene expression, as assessed by the SRE.L reporter gene, is synergistically activated by Galpha(q) and Rho GEFs. The synergistic effect of Galpha(q) on this response is inhibited by C3 exoenzyme and requires phospholipase C activation. Surprisingly, expression of Galpha(q), in contrast to that of Galpha(12) and Galpha(13), does not increase the amount of activated Rho. We also observe that Galpha(q) enhances SRE.L stimulation by activated Rho, indicating that the effect of Galpha(q) occurs downstream of Rho activation. Thus, Galpha(q) interacts physically and/or functionally with Rho GEFs; however this does not appear to lead to or result from increased activation of Rho. We suggest that Galpha(q)-generated signals enhance responses downstream of Rho activation.

    Funded by: NCI NIH HHS: CA62029; NIAID NIH HHS: AI38396, R01 AI038396, R29 AI038396; NIGMS NIH HHS: GM36927, R01 GM036927, R56 GM036927

    The Journal of biological chemistry 2001;276;18;15445-52

  • Characterization of RGS5 in regulation of G protein-coupled receptor signaling.

    Zhou J, Moroi K, Nishiyama M, Usui H, Seki N, Ishida J, Fukamizu A and Kimura S

    Department of Biochemistry and Molecular Pharmacology, Chiba University Graduate School of Medicine, Japan.

    RGS proteins (regulators of G protein signaling) serve as GTPase-activating proteins (GAPs) for G alpha subunits and negatively regulate G protein-coupled receptor signaling. In this study, we characterized biochemical properties of RGS5 and its N terminal (1-33)-deleted mutant (deltaN-RGS5). RGS5 bound to G alpha(i1), G alpha(i2), G alpha(i3), G alpha(o) and G alpha(q) but not to G alpha(s) and G alpha13 in the presence of GDP/AIF4-, and accelerated the catalytic rate of GTP hydrolysis of G alpha(i3) subunit. When expressed in 293T cells stably expressing angiotensin (Ang) AT1a receptors (AT1a-293T cells), RGS5 suppressed Ang II- and endothelin (ET)-1-induced intracellular Ca2+ transients. The effect of RGS5 was concentration-dependent, and the slope of the concentration-response relationship showed that a 10-fold increase in amounts of RGS5 induced about 20-25% reduction of the Ca2+ signaling. Furthermore, a comparison study of three sets of 293T cells with different expression levels of AT1a receptors showed that RGS5 inhibited Ang II-induced responses more effectively in 293T cells with the lower density of AT1a receptors, suggesting that the degree of inhibition by RGS proteins reflects the ratio of amounts of RGS proteins to those of activated G alpha subunits after receptor stimulation by agonists. When expressed in AT1a-293T cells, deltaN-RGS5 was localized almost exclusively in the cytosolic fraction, and exerted the inhibitory effects as potently as RGS5 which was present in both membrane and cytosolic fractions. Studies on relationship between subcellular localization and inhibitory effects of RGS5 and deltaN-RGS5 revealed that the N terminal (1-33) of RGS5 plays a role in targeting this protein to membranes, and that the N terminal region of RGS5 is not essential for exerting activities.

    Life sciences 2001;68;13;1457-69

  • Cloning and characterization of the human phosphoinositide-specific phospholipase C-beta 1 (PLC beta 1).

    Caricasole A, Sala C, Roncarati R, Formenti E and Terstappen GC

    Biology Department, GlaxoWellcome Medicines Research Centre, Verona, Italy.

    Phospholipase C-beta (PLC beta) catalyses the generation of inositol 1,4,5-trisphosphate (IP(3)) and diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate (IP(2)), a key step in the intracellular transduction of a large number of extracellular signals, including neurotransmitters and hormones modulating diverse developmental and functional aspects of the mammalian central nervous system. Four mammalian isozymes are known (PLC beta 1-4), which differ in their function and expression patterns in vivo. We have characterized the human PLC beta 1 genomic locus (PLC beta 1), cloned two distinct PLC beta 1 cDNAs (PLC beta 1a and b) and analysed their respective expression patterns in a comprehensive panel of human tissues using quantitative TaqMan technology. The two cDNAs derive from transcripts generated through alternative splicing at their 3' end, and are predicted to encode for PLC beta 1 isoforms differing at their carboxy-terminus. The human PLC beta 1 isoforms are co-expressed in the same tissues with a distinctly CNS-specific profile of expression. Quantitative differences in PLC beta 1 isoform expression levels are observed in some tissues. Transient expression of epitope-tagged versions of the two isoforms followed by immunofluorescence revealed localization of the proteins to the cytoplasm and the inner side of the cell membrane. Finally, we characterized the structure of the PLC beta 1 locus and confirmed its mapping to human chromosome 20.

    Biochimica et biophysica acta 2000;1517;1;63-72

  • DNA cloning using in vitro site-specific recombination.

    Hartley JL, Temple GF and Brasch MA

    Life Technologies, Inc., Rockville, Maryland 20850, USA. jhartley@lifetech.com

    As a result of numerous genome sequencing projects, large numbers of candidate open reading frames are being identified, many of which have no known function. Analysis of these genes typically involves the transfer of DNA segments into a variety of vector backgrounds for protein expression and functional analysis. We describe a method called recombinational cloning that uses in vitro site-specific recombination to accomplish the directional cloning of PCR products and the subsequent automatic subcloning of the DNA segment into new vector backbones at high efficiency. Numerous DNA segments can be transferred in parallel into many different vector backgrounds, providing an approach to high-throughput, in-depth functional analysis of genes and rapid optimization of protein expression. The resulting subclones maintain orientation and reading frame register, allowing amino- and carboxy-terminal translation fusions to be generated. In this paper, we outline the concepts of this approach and provide several examples that highlight some of its potential.

    Genome research 2000;10;11;1788-95

  • Alterations in Ca2+ cycling proteins and G alpha q signaling after left ventricular assist device support in failing human hearts.

    Takeishi Y, Jalili T, Hoit BD, Kirkpatrick DL, Wagoner LE, Abraham WT and Walsh RA

    Department of Medicine, Case Western Reserve University, Cleveland, OH, USA.

    Objective: Left ventricular assist device support mechanically unloads the failing ventricle with resultant improvement in cardiac geometry and function in patients with end-stage heart failure. Activation of the G alpha q signaling pathway, including protein kinase C, appears to be involved in the progression of heart failure. Similarly down-regulation of Ca2+ cycling proteins may contribute to contractile depression in this clinical syndrome. Thus we examined whether protein kinase C activation and decreased Ca2+ cycling protein levels could be reversed by left ventricular assist device support.

    Methods: Left ventricular myocardial specimens were obtained from seven patients during placement of left ventricular assist device and heart transplantation. We examined changes in protein levels of G alpha q, phospholipase C beta 1, regulators of G protein signaling (RGS), sarcoplasmic reticulum Ca2+ ATPase, phospholamban and translocation of protein kinase C isoforms (alpha, beta 1, and beta 2).

    Results: The paired pre- and post-left ventricular assist device samples revealed that RGS2, a selective inhibitor of G alpha q, was decreased (P < 0.01), while the status of G alpha q, phospholipase C beta 1, RGS3 and RGS4 were unchanged after left ventricular assist device implantation. Translocation of protein kinase C isoforms remained unchanged. Left ventricular assist device support increased sarcoplasmic reticulum Ca2+ ATPase protein level (P < 0.01), while phospholamban abundance was unchanged.

    Conclusions: We conclude that altered protein expression and stoichiometry of the major cardiomyocyte Ca2+ cycling proteins rather than reduced phospholipase C beta 1 activation may contribute to improved mechanical function produced by left ventricular assist device support in human heart failure.

    Funded by: NHLBI NIH HHS: P50 HL52318

    Cardiovascular research 2000;45;4;883-8

  • Identification of G-protein coupled receptor subunits in normal human dental pulp.

    Pozo D, Segura JJ, Jiménez-Rubio A, García-Pergañeda A, Bettahi I, Guerrero JM and Calvo JR

    Department of Medical Biochemistry and Molecular Biology, School of Medicine and Dentistry, University of Seville, Avda. Sánchez Pizjuan, 4, 41009 Seville, Spain.

    To respond appropriately to their environment, dental pulp cells must integrate informational input from multiple ligands, such as neuropeptides, growth factors, and vasoactive amines. These ligands act through multiple intracellular signaling pathways. G-protein coupled receptor subunits play a major role in this process, providing a mechanism for coordinated regulation of both messengers and effectors. Increasing number of neuropeptides have been found in pulpal tissue. However, there is no data about molecular identification of G-protein subunits in human dental pulp. To identify the postreceptor mechanism involved in dental pulp cell signal transduction, we performed a Western blot analysis of different G-protein subunits. Biopsy specimens of human dental pulp were prepared and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by analysis with appropriate antibodies. We detected G alpha q/alpha 11, short and long forms of G alpha s, beta common, Gio-3, and Gil-2 antigens with a molecular weight approximately 42 kDa, 42 and 45 kDa, 36 kDa, 40 kDa, and 40 kDa, respectively. These results indicate that human pulp cells possess the cellular machinery to respond to sensory neuropeptides when they are released from the peptidergic nerve fibers. On this basis, the relationships of postdevelopmental, age-dependent, and pathophysiological disorders of G-proteins subunits in dental pulp could be studied.

    Journal of endodontics 2000;26;1;16-9

  • Interaction of folate receptor with signaling molecules lyn and G(alpha)(i-3) in detergent-resistant complexes from the ovary carcinoma cell line IGROV1.

    Miotti S, Bagnoli M, Tomassetti A, Colnaghi MI and Canevari S

    Unit of Molecular Therapies, Department of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy. miotti@istitutotumori.mi.it.

    Using as a model the ovary carcinoma cell line IGROV1, we analyzed the partitioning of the glycosyl-phosphatidylinositol-anchored folate receptor into lipid rafts based on its relative detergent insolubility, with a focus on physically and functionally associated signaling molecules. A variable amount (40-60%) of folate receptor was found in low-density Triton X-100 insoluble complexes together with subunits of heterotrimeric G-proteins and the src-family non-receptor tyrosine kinases p53-56 lyn. In the same fraction the structural component of caveolae, caveolin, was not detected at the protein level, although the corresponding mRNA was detected in trace amounts. Comodulation of folate receptor and signalling molecules was observed in the detergent-insoluble complexes during cell proliferation or induced by phosphatidylinositol-specific phospholipase C treatment or by interaction with anti-folate receptor monoclonal antibodies. Moreover, complexes of folate receptor, lyn and the G(&agr;)(i-3) subunit were immunoprecipitated using either anti-folate receptor or anti-lyn antibodies. In vitro kinase assay of the immunoprecipitates revealed stimulation of phosphorylation of common and specific proteins. In particular, the p53 form of lyn appeared to be enriched and phosphorylated in the anti-folate receptor MOv19 monoclonal antibody immunoprecipitate, whereas a 40 kDa band common to anti-folate receptor and anti-lyn immunoprecipitates was the phosphorylated form of the G(&agr;)(i-3) subunit. These findings point to the functional interaction between folate receptor and associated signaling molecules.

    Journal of cell science 2000;113 Pt 2;349-57

  • A novel spliced variant of the type 1 corticotropin-releasing hormone receptor with a deletion in the seventh transmembrane domain present in the human pregnant term myometrium and fetal membranes.

    Grammatopoulos DK, Dai Y, Randeva HS, Levine MA, Karteris E, Easton AJ and Hillhouse EW

    Sir Quinton Hazell Molecular Medicine Research Centre, Department of Biological Sciences, University of Warwick, Coventry, United Kingdom. chdg@dna.bio.warwick.ac.uk

    CRH exerts its actions via activation of specific G protein-coupled receptors, which exist in two types, CRH-R1 and CRH-R2, and arise from different genes with multiple spliced variants. RT-PCR amplification of CRH receptor sequences from human myometrium and fetal membranes yielded cDNAs that encode a novel CRH-R type 1 spliced variant. This variant (CRH-R1d) is present in the human pregnant myometrium at term only, which suggests a physiologically important role at the end of human pregnancy and labor. The amino acid sequence of CRH-R1d is identical to the CRH-R1alpha receptor except that it contains an exon deletion resulting in the absence of 14 amino acids in the predicted seventh transmembrane domain. Binding studies in HEK-293 cells stably expressing the CRH-R1d or CRH-R1alpha receptors revealed that the deletion does not change the binding characteristics of the variant receptor. In contrast, studies on the G protein activation demonstrated that CRH-R1d is not well coupled to the four subtypes of G proteins (G(s), G(i), G(o), G(q)) that CRH-R1alpha can activate. These data suggest that although the deleted segment is not important for CRH binding, it plays a crucial role in CRH receptor signal transduction. Second messenger studies of the variant receptor showed that CRH and CRH-like peptides can stimulate the adenylate cyclase system, with reduced sensitivity and potency by 10-fold compared with the CRH-R1alpha. Furthermore, CRH failed to stimulate inositol trisphosphate production. Coexpression studies between the CRH-R1d or CRH-R1alpha showed that this receptor does not play a role as a dominant negative receptor for CRH.

    Funded by: Wellcome Trust

    Molecular endocrinology (Baltimore, Md.) 1999;13;12;2189-202

  • Calmodulin dependence of presynaptic metabotropic glutamate receptor signaling.

    O'Connor V, El Far O, Bofill-Cardona E, Nanoff C, Freissmuth M, Karschin A, Airas JM, Betz H and Boehm S

    Department of Neurochemistry, Max Planck Institute for Brain Research, Deutschordenstrasse 46, 60528 Frankfurt, Germany.

    Glutamatergic neurotransmission is controlled by presynaptic metabotropic glutamate receptors (mGluRs). A subdomain in the intracellular carboxyl-terminal tail of group III mGluRs binds calmodulin and heterotrimeric guanosine triphosphate-binding protein (G protein) betagamma subunits in a mutually exclusive manner. Mutations interfering with calmodulin binding and calmodulin antagonists inhibit G protein-mediated modulation of ionic currents by mGluR 7. Calmodulin antagonists also prevent inhibition of excitatory neurotransmission via presynaptic mGluRs. These results reveal a novel mechanism of presynaptic modulation in which Ca(2+)-calmodulin is required to release G protein betagamma subunits from the C-tail of group III mGluRs in order to mediate glutamatergic autoinhibition.

    Science (New York, N.Y.) 1999;286;5442;1180-4

  • Differential coupling of the sphingosine 1-phosphate receptors Edg-1, Edg-3, and H218/Edg-5 to the G(i), G(q), and G(12) families of heterotrimeric G proteins.

    Windh RT, Lee MJ, Hla T, An S, Barr AJ and Manning DR

    Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

    Sphingosine 1-phosphate (S1P) is one of several bioactive phospholipids that exert profound mitogenic and morphogenic actions. Originally characterized as a second messenger, S1P is now recognized to achieve many of its effects through cell surface, G protein-coupled receptors. We used a subunit-selective [(35)S]GTPgammaS binding assay to investigate whether the variety of actions exerted through Edg-1, a recently identified receptor for S1P, might be achieved through multiple G proteins. We found, employing both Sf9 and HEK293 cells, that Edg-1 activates only members of the G(i) family, and not G(s), G(q), G(12), or G(13). We additionally established that Edg-1 activates G(i) in response not only to S1P but also sphingosylphosphorylcholine; no effects of lysophosphatidic acid through Edg-1 were evident. Our assays further revealed a receptor(s) for S1P endogenous to HEK293 cells that mediates activation of G(13) as well as G(i). Because several of the biological actions of S1P are assumed to proceed through the G(12/13) family, we tested whether Edg-3 and H218/Edg-5, two other receptors for S1P, might have a broader coupling profile than Edg-1. Indeed, Edg-3 and H218/Edg-5 communicate not only with G(i) but also with G(q) and G(13). These studies represent the first characterization of S1P receptor activity through G proteins directly and establish fundamental differences in coupling.

    Funded by: NIGMS NIH HHS: GM51196

    The Journal of biological chemistry 1999;274;39;27351-8

  • The prostacyclin receptor is isoprenylated. Isoprenylation is required for efficient receptor-effector coupling.

    Hayes JS, Lawler OA, Walsh MT and Kinsella BT

    Department of Biochemistry, Merville House, University College Dublin, Belfield, Dublin 4, Ireland.

    The prostacyclin receptor (IP), a G protein-coupled receptor, mediates the actions of the prostanoid prostacyclin and its mimetics. IPs from a number of species each contain identically conserved putative isoprenylation CAAX motifs, each with the sequence CSLC. Metabolic labeling of human embryonic kidney (HEK) 293 cells stably overexpressing the hemagluttinin epitope-tagged IP in the presence of [(3)H]mevalonolactone established that the mouse IP is isoprenylated. Studies involving in vitro assays confirmed that recombinant forms of the human and mouse IP are modified by carbon 15 farnesyl isoprenoids. Disruption of isoprenylation, by site-directed mutagenesis of Cys(414) to Ser(414), within the CAAX motif, abolished isoprenylation of IP(SSLC) both in vitro and in transfected cells. Scatchard analysis of the wild type (IP) and mutant (IP(SSLC)) receptor confirmed that each receptor exhibited high and low affinity binding sites for [(3)H]iloprost, which were not influenced by receptor isoprenylation. Whereas stable cell lines overexpressing IP generated significant agonist (iloprost and cicaprost)-mediated increases in cAMP relative to nontransfected cells, cAMP generation by IP(SSLC) cells was not significantly different from the control, nontransfected HEK 293 cells. Moreover, co-expression of the alpha (alpha) subunit of Gs generated significant augmentations in cAMP by IP but not by IP(SSLC) cells. Whereas IP also demonstrated significant, dose-dependent increases in [Ca(2+)](i) in response to iloprost or cicaprost compared with the nontransfected HEK 293 cells, mobilization of [Ca(2+)](i) by IP(SSLC) was significantly impaired. Co-transfection of cells with either Galpha(q) or Galpha(11) resulted in significant augmentation of agonist-mediated [Ca(2+)](i) mobilization by IP cells but not by IP(SSLC) cells or by the control, HEK 293 cells. In addition, inhibition of isoprenylation by lovastatin treatment significantly reduced agonist-mediated cAMP generation by IP in comparison to the nonisoprenylated beta(2) adrenergic receptor or nontreated cells. Hence, isoprenylation of IP does not influence ligand binding but is required for efficient coupling to the effectors adenylyl cyclase and phospholipase C.

    The Journal of biological chemistry 1999;274;34;23707-18

  • Identification of the binding site for Gqalpha on its effector Bruton's tyrosine kinase.

    Ma YC and Huang XY

    Department of Physiology, Cornell University Medical College, 1300 York Avenue, New York, NY 10021, USA.

    Heterotrimeric G proteins and tyrosine kinases are two major cellular signal transducers. Although G proteins are known to activate tyrosine kinases, the activation mechanism is not clear. Here, we demonstrate that G protein Gqalpha binds directly to the nonreceptor Bruton's tyrosine kinase (Btk) to a region composed of a Tec-homology (TH) domain and a sarcoma virus tyrosine kinase (Src)-homology 3 (SH3) domain both in vitro and in vivo. Only active GTP-bound Gqalpha, not inactive GDP-bound Gqalpha, can bind to Btk. Mutations of Btk that disrupt its ability to bind Gqalpha also eliminate Btk stimulation by Gqalpha, suggesting that this interaction is important for Btk activation. Remarkably, the structure of this TH (including a proline-rich sequence) -SH3 fragment of the Btk family of tyrosine kinases shows an intramolecular interaction. Furthermore, the crystal structure of the Src family of tyrosine kinases reveals that the intramolecular interaction of SH3 and its ligand is the major determining factor keeping the kinase inactive. Thus, we propose an activation model that entails binding of Gqalpha to the TH-SH3 region, thereby disrupting the TH-SH3 intramolecular interaction and activating Btk.

    Proceedings of the National Academy of Sciences of the United States of America 1998;95;21;12197-201

  • Expression of GTPase-deficient Gialpha2 results in translocation of cytoplasmic RGS4 to the plasma membrane.

    Druey KM, Sullivan BM, Brown D, Fischer ER, Watson N, Blumer KJ, Gerfen CR, Scheschonka A and Kehrl JH

    Laboratory of Immunoregulation, National Institutes of Health, Bethesda, Maryland 20892-1876, USA.

    The members of a recently identified protein family termed regulators of G-protein signaling (RGS) act as GTPase-activating proteins for certain Galpha subunits in vitro, but their physiological effects in cells are uncertain in the face of similar biochemical activity and overlapping patterns of tissue expression. Consistent with its activity in in vitro GTPase-activating protein assays, RGS4 interacts efficiently with endogenous proteins of the Gi and Gq subclasses of Galpha subunits but not with G12alpha or Gsalpha. Unlike other RGS proteins such as RGS9, RGS-GAIP, and Sst2p, which have been reported to be largely membrane-associated, a majority of cellular RGS4 is found as a soluble protein in the cytoplasm. However, the expression of a GTPase-deficient Gialpha subunit (Gialpha2-Q204L) resulted in the translocation of both wild type RGS4 and a non-Gialpha-binding mutant (L159F) to the plasma membrane. These data suggest that RGS4 may be recruited to the plasma membrane indirectly by G-protein activation and that multiple RGS proteins within a given cell might be differentially localized to determine a physiologic response to a G-protein-linked stimulus.

    Funded by: NIDDK NIH HHS: DK38452

    The Journal of biological chemistry 1998;273;29;18405-10

  • Molecular characterization, pharmacological properties and chromosomal localization of the human GALR2 galanin receptor.

    Fathi Z, Battaglino PM, Iben LG, Li H, Baker E, Zhang D, McGovern R, Mahle CD, Sutherland GR, Iismaa TP, Dickinson KE and Zimanyi IA

    Neuroscience Drug Discovery, Bristol-Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway, Wallingford, CT 06492, USA. fathi@bms.com

    The neuropeptide galanin mediates a diverse spectrum of biological activities by interacting with specific G protein-coupled receptors. We have used homology genomic library screening and polymerase chain reaction (PCR) techniques to isolate both genomic and cDNA clones encoding the human homolog of the recently cloned rat GALR2 galanin receptor. By fluorescence in situ hybridization, the gene encoding human GALR2 (GALNR2) has been localized to chromosome 17q25.3. The two coding exons of the human GALNR2 gene, interrupted by an intron positioned at the end of transmembrane domain III, encode a 387 amino acid G protein-coupled receptor with 87% overall amino acid identity with rat GALR2. In HEK-293 cells stably expressing human GALR2, binding of [125I]porcine galanin is saturable and can be displaced by galanin, amino-terminal galanin fragments and chimeric galanin peptides but not by carboxy-terminal galanin fragments. In HEK-293 cells, human GALR2 couples both to Galphaq/11 to stimulate phospholipase C and increase intracellular calcium levels and to Galphai/o to inhibit forskolin-stimulated intracellular cAMP accumulation. A wide tissue distribution is observed by reverse transcriptase (RT)-PCR analysis, with human GALR2 mRNA being detected in many areas of the human central nervous system as well as in peripheral tissues.

    Brain research. Molecular brain research 1998;58;1-2;156-69

  • G alpha q cDNA sequence from human platelets.

    Gabbeta J, Dhanasekaran N and Rao AK

    Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140, USA.

    G-proteins play a major role in cell signaling and specific abnormalities in these proteins have been implicated in some diseases. Although, two human G alpha q coding sequences are published, they differ at functionally important regions. We have sequenced the human G alpha q cDNA synthesized from normal platelet mRNA by reverse transcription. Our results showed that the penultimate amino acid leucine is conserved in the human G alpha q sequence which has 94.4% identity in nucleotide sequence and 99.2% homology in amino acid sequence with murine sequence.

    Funded by: NHLBI NIH HHS: K077HL02658; NIGMS NIH HHS: GM 49897

    Thrombosis research 1998;91;1;29-32

  • Role of the second and third intracellular loops of metabotropic glutamate receptors in mediating dual signal transduction activation.

    Francesconi A and Duvoisin RM

    Margaret M. Dyson Vision Research Institute, Department of Ophthalmology, Cornell University Medical College, New York, New York 10021, USA. afrance@med.cornell.edu

    On the basis of sequence homology and structural similarities, metabotropic glutamate receptors (mGluRs), extracellular Ca2+-sensing receptor, gamma-aminobutyric acid type B receptor, and pheromone receptors are enlisted in a distinct family within the larger G protein-coupled receptor superfamily. When expressed in heterologous systems, group I mGluRs can activate dual signal transduction pathways, phosphoinositides turnover and cAMP production. To investigate the structural basis of these coupling properties, we introduced single amino acid substitutions within the second and third intracellular loops (i2 and i3) of mGluR1alpha. Wild-type and mutant receptors were expressed in human embryonic kidney 293 cells and analyzed for their capacity to stimulate both signaling cascades. Each domain appeared to be critical for the coupling to phospholipase C and adenylyl cyclase. Within i2, Thr695, Lys697, and Ser702 were found to be selectively involved in the interaction with Gq class alpha subunit(s), whereas mutation of Pro698 and the deletion Cys694-Thr695 affected only Gs coupling. Furthermore, the mutation K690A profoundly altered mGluR1alpha signaling properties and imparted to the receptor the ability to couple to the inhibitory cAMP pathway. Within i3, we uncovered two residues, Arg775 and Phe781, that are crucial for coupling to both pathways, since their substitution leads to receptor inactivation.

    Funded by: NEI NIH HHS: EY09534

    The Journal of biological chemistry 1998;273;10;5615-24

  • Evidence for the involvement of several intracellular domains in the coupling of oxytocin receptor to G alpha(q/11).

    Qian A, Wang W and Sanborn BM

    Department of Biochemistry and Molecular Biology, University of Texas Houston Medical School 77030, USA.

    In order to probe the nature of oxytocin receptor (OTR)/G alpha(q/11) protein coupling, we examined the effect of co-expression of OTR intracellular domains on oxytocin-stimulated phosphoinositide turnover in COSM6 cells overexpressing OTR and G alpha(q). Co-expression of G alpha(q) enhanced the oxytocin response maximally at a pOTR/pG alpha(q) plasmid transfection ratio of 1:0.16. In cells co-expressing OTR and G alpha(q/11), oxytocin stimulated phosphoinositide turnover with an EC50 of 48 nM. Co-transfection with plasmids expressing OTR intracellular domains inhibited oxytocin-stimulated phosphoinositide turnover by 23 +/- 6% (1i), 37 +/- 4% (2i), 55 +/- 6% (3i), and 40 +/- 6% (4i), respectively (P < 0.01). Expression of the 3i loop of the alpha(1B)-adrenergic receptor, which also couples to G alpha(q/11), inhibited phosphoinositide turnover by 35 +/- 2% (P < 0.01), while expression of the 3i loop of the dopamine 1A receptor, which couples to G alpha(s), had no effect. While these data indicate a functional role for the OTR 3i loop, they also suggest that interactions with more than one intracellular domain probably mediate the coupling of OTR to the G alpha(q/11) class of GTP-binding proteins.

    Funded by: NICHD NIH HHS: HD09618, T32-HD07324

    Cellular signalling 1998;10;2;101-5

  • RGS2/G0S8 is a selective inhibitor of Gqalpha function.

    Heximer SP, Watson N, Linder ME, Blumer KJ and Hepler JR

    Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA.

    RGS (regulators of G protein signaling) proteins are GTPase activating proteins that inhibit signaling by heterotrimeric G proteins. All RGS proteins studied to date act on members of the Gialpha family, but not Gsalpha or G12alpha. RGS4 regulates Gialpha family members and Gqalpha. RGS2 (G0S8) is exceptional because the G proteins it regulates have not been identified. We report that RGS2 is a selective and potent inhibitor of Gqalpha function. RGS2 selectively binds Gqalpha, but not other Galpha proteins (Gi, Go, Gs, G12/13) in brain membranes; RGS4 binds Gqalpha and Gialpha family members. RGS2 binds purified recombinant Gqalpha, but not Goalpha, whereas RGS4 binds either. RGS2 does not stimulate the GTPase activities of Gsalpha or Gialpha family members, even at a protein concentration 3000-fold higher than is sufficient to observe effects of RGS4 on Gialpha family members. In contrast, RGS2 and RGS4 completely inhibit Gq-directed activation of phospholipase C in cell membranes. When reconstituted with phospholipid vesicles, RGS2 is 10-fold more potent than RGS4 in blocking Gqalpha-directed activation of phospholipase Cbeta1. These results identify a clear physiological role for RGS2, and describe the first example of an RGS protein that is a selective inhibitor of Gqalpha function.

    Funded by: NIAMS NIH HHS: AR-07279-18, T32 AR007279

    Proceedings of the National Academy of Sciences of the United States of America 1997;94;26;14389-93

  • Platelet signal transduction defect with Galpha subunit dysfunction and diminished Galphaq in a patient with abnormal platelet responses.

    Gabbeta J, Yang X, Kowalska MA, Sun L, Dhanasekaran N and Rao AK

    The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140, USA.

    G proteins play a major role in signal transduction upon platelet activation. We have previously reported a patient with impaired agonist-induced aggregation, secretion, arachidonate release, and Ca2+ mobilization. Present studies demonstrated that platelet phospholipase A2 (cytosolic and membrane) activity in the patient was normal. Receptor-mediated activation of glycoprotein (GP) IIb-IIIa complex measured by flow cytometry using antibody PAC-1 was diminished despite normal amounts of GPIIb-IIIa on platelets. Ca2+ release induced by guanosine 5'-[gamma-thio]triphosphate (GTP[gammaS]) was diminished in the patient's platelets, suggesting a defect distal to agonist receptors. GTPase activity (a function of alpha-subunit) in platelet membranes was normal in resting state but was diminished compared with normal subjects on stimulation with thrombin, platelet-activating factor, or the thromboxane A2 analog U46619. Binding of 35S-labeled GTP[gammaS] to platelet membranes was decreased under both basal and thrombin-stimulated states. Iloprost (a stable prostaglandin I2 analog) -induced rise in cAMP (mediated by Galphas) and its inhibition (mediated by Galphai) by thrombin in the patient's platelet membranes were normal. Immunoblot analysis of Galpha subunits in the patient's platelet membranes showed a decrease in Galphaq (<50%) but not Galphai, Galphaz, Galpha12, and Galpha13. These studies provide evidence for a hitherto undescribed defect in human platelet G-protein alpha-subunit function leading to impaired platelet responses, and they provide further evidence for a major role of Galphaq in thrombin-induced responses.

    Funded by: NHLBI NIH HHS: K077HL02658, T32 HL007777, T32 HL07777; NIGMS NIH HHS: GM 49897, R01 GM049897

    Proceedings of the National Academy of Sciences of the United States of America 1997;94;16;8750-5

  • Signaling through G proteins in platelets: to the integrins and beyond.

    Brass LF, Manning DR, Cichowski K and Abrams CS

    Department of Medicine, University of Pennsylvania, Philadelphia 19104, USA. brass@mail.med.upenn.edu

    Many of the agonists that cause platelet activation are thought to do so by interacting with G protein-coupled receptors on the platelet surface. By activating heterotrimeric G proteins, these receptors evoke shape change, granule secretion and platelet aggregation. This review provides a brief overview of these events, summarizes current information about the role of pleckstrin in events downstream from G protein-coupled receptors, and briefly considers the signaling pathways that couple G protein activation to the low molecular weight GTP-binding proteins which control cytoskeletal reorganization and fibrinogen receptor exposure during platelet activation.

    Funded by: NHLBI NIH HHS: HL40387, HL45181, HL54500; ...

    Thrombosis and haemostasis 1997;78;1;581-9

  • Interaction of the G-protein G11alpha with receptors and phosphoinositidase C: the contribution of G-protein palmitoylation and membrane association.

    Wise A, Parenti M and Milligan G

    Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Scotland, UK.

    Wild-type and palmitoylation defective mutants of the murine G protein G11alpha were transfected into HEK293 cells. Wild-type G11alpha was membrane associated, Cys9Ser Cys10Ser G11alpha was present in the soluble fraction whilst both Cys9Ser G11alpha and Cys10Ser G11alpha were distributed between the fractions. Expression of the rat TRH receptor resulted in agonist stimulation of inositol phosphate accumulation. The degree of stimulation produced by TRH following co-transfection of the palmitoylation-resistant forms of G11alpha compared to the wild-type protein correlated with the amount of membrane-associated G protein.

    Funded by: Wellcome Trust

    FEBS letters 1997;407;3;257-60

  • The human thromboxane A2 receptor alpha isoform (TP alpha) functionally couples to the G proteins Gq and G11 in vivo and is activated by the isoprostane 8-epi prostaglandin F2 alpha.

    Kinsella BT, O'Mahony DJ and Fitzgerald GA

    Department of Biochemistry, University College Dublin, Ireland.

    To establish whether the thromboxane A2 (TXA2) receptor (TP) functionally couples to the Gq family of heterotrimeric G proteins in vivo, we have coexpressed the cDNAs coding for the human platelet/placental TP alpha isoform (TP alpha) and the alpha subunits of Gq or G11 in human embryonic kidney (HEK) 293 cells. TP activation in response to ligand stimulation was monitored by analyzing mobilization of intracellular calcium (Ca++i) in FURA2/AM-loaded transfected HEK 293 and in platelets. Second, we wished to examine the possible interaction of the isoprostane 8-epi prostaglandin F2 alpha with the TP alpha, in transfected HEK 293 cells and with the TPs expressed in platelets. Thus both the prostaglandin endoperoxide/TXA2 analog (U46619) and the 8-epi PGF2 alpha were utilized as ligand probes of TP alpha activation. The results demonstrate that each ligand induced elevations of Ca++i levels in HEK 293 cells, cotransfected with either the TP alpha and G alpha q or the TP alpha and G alpha 11, and also in platelets. Initial stimulation of these cells with U46619 or 8-epi PGF 2 alpha desensitized a subsequent rise in [Ca++]i in response to U46619 or 8-epi PGF 2 alpha, respectively. Moreover, prestimulation with U46619 desensitized a subsequent rise in Ca++i concentration in response to 8-epi PGF 2 alpha, and vice versa. These responses were blocked by the TP antagonist SQ29,548 in both cell types. In contrast, prestimulation of the transfected HEK 293 cells or platelets with thrombin did not desensitize a subsequent rise in [Ca++]i in response to U46619 or 8-epi PGF 2 alpha. After stimulation with either U46619 or 8-epi PGF 2 alpha, no significant rise in Ca++i levels was observed in HEK 293 cells transfected with the TP alpha receptor only or in control cells transfected with the vector pCMV5. These results demonstrate that the TP alpha isoform functionally couples with either Gq or G11 in vivo, whether activated by a PG/TXA2 analog or by the F2 isoprostane 8-epi PGF2 alpha.

    Funded by: Wellcome Trust

    The Journal of pharmacology and experimental therapeutics 1997;281;2;957-64

  • Expression of G proteins in human placentas from pregnancies complicated by gestational hypertension.

    Petit A, Geoffroy P and Bélisle S

    Department of Obstetrics and Gynecology, University of Montréal, Research Center, Ste-Justine Hospital, Québec, Canada.

    Preeclampsia (gestational hypertension) is accompanied by decreased hPL and increased hCG levels in maternal serum. The expression of these peptides as well as the endocrine mechanisms responsible for their regulation in preeclampsia are unknown. We have demonstrated that regulatory GTP-binding proteins (G proteins) are implicated in the modulation of hPL production by placentas from normal pregnancies. In order to extend our knowledge on placental endocrinology, we analyzed in this study the expression of hPL and beta-hCG mRNAs as well as placental G protein alpha-subunits in pregnancies complicated by gestational hypertension. Western and Northern blot analyses were respectively performed on membrane protein and total mRNA preparations from human placentas of preeclamptic (n = 7) and normal pregnancies (n = 4). The levels of hPL and beta-hCG mRNAs were respectively 108% and 105% of those from normal placentas, suggesting that the altered circulating levels of hPL and beta-hCG are not related to dysfunctional mRNA expression of these peptides. The autoradiographs for G proteins and their mRNAs showed no difference in G protein expression between preeclamptic and normal tissues. Specifically, G alpha i2, G alpha i3, G alpha o, G alpha s, and G alpha q/11 levels reached 87%, 81%, 91%, 99%, and 103% respectively of those from normal placentas. In parallel with the protein levels, their mRNAs expression were respectively 93%, 89%, 113%, 104%, and 94% of normal values for G alpha i2, G alpha i3, G alpha o, G alpha s, and G alpha q/11. These results suggest that neither a change in hPL and beta-hCG expression nor a change in signal transduction machinery is implicated in the altered circulating levels of hPL and beta-hCG in preeclampsia.

    Life sciences 1997;60;12;953-60

  • Expression of angiotensin II type-I receptor and phospholipase C-linked G alpha q/11 protein in the human placenta.

    Petit A, Geoffroy P and Bélisle S

    Department of Obstetrics and Gynecology, University of Montréal, Research Center, Ste-Justine Hospital, Québec, Canada.

    Objective: A recent study on the distribution of angiotensin II (ANG II), ANG II (AT) receptors, and human placental lactogen in human placental tissues from term pregnancies showed positive correlations among these indices, suggesting an important role for ANG II in placental endocrinology. However, nothing is known about the ontogenesis of this functional role for ANG II during pregnancy. Therefore, the aim of this study was to investigate the placental AT receptor expression at various trimesters of pregnancy. We also studied the ontogenesis of phospholipase C-linked G alpha q/11 protein, which is known to transduce ANG II type-1 (AT1) receptor signal.

    Methods: Western blot analysis of placental membrane proteins was performed using a chemiluminescence kit and specific antibodies against AT1 receptor and G alpha q/11 protein. Northern blot analyses of AT1 receptor and G alpha q/11 mRNA expression were accomplished using random primed [32P]dCTP-labeled specific probes.

    Results: The autoradiographs of AT1 receptor mRNAs (2.4 kb) and proteins (83 kDa) showed a progressive 4.8-fold and 2.6-fold increase, respectively, during pregnancy, with maximal levels at term. We also observed progressive 1.8-fold and 4.5-fold increases of G alpha q/11 protein (43 kDa) and mRNAs (4.5, 6.0, and 7.1 kb), respectively, during pregnancy, with maximal levels observed at term.

    Conclusion: Our results demonstrate that both the human placental AT1 receptor content and the G alpha q/11 protein level increase during pregnancy, and suggest that the AT1 receptor pathway may play a role in human placental physiology.

    Journal of the Society for Gynecologic Investigation 1996;3;6;316-21

  • Coupling of metabotropic glutamate receptors 2 and 4 to G alpha 15, G alpha 16, and chimeric G alpha q/i proteins: characterization of new antagonists.

    Gomeza J, Mary S, Brabet I, Parmentier ML, Restituito S, Bockaert J and Pin JP

    UPR Centre Nationale de Recherche Scientifique 9023, Mécanismes Moléculaires des Communications Cellulaires, CCIPE, Montpeller, France.

    Together with the calcium-sensing receptor, the metabotropic glutamate receptors (mGluRs) share no sequence homology with the other G protein-coupled receptors (GPCRs) and therefore constitute a new family of receptors. Recently, it was reported that G alpha 15 and G alpha 16 subunits allow many GPCRs to activate phospholipase C (PLC). Furthermore, the exchange of a few carboxyl-terminal residues of G alpha q by those of G alpha 12 or G alpha o allows the resulting chimeric G alpha subunits (G alpha ql and G alpha qol respectively) to couple Gi-coupled receptors to PLC. We report that mGluR2 and mGluR4, two receptors negatively coupled to adenylyl cyclase, activate PLC when coexpressed with G alpha 15, G alpha ql or G alpha qo. This indicates that the carboxyl-terminal end of the G alpha subunit also plays an important role in the specific interaction between mGluRs and the G proteins. In addition, the measurement of PLC activation by Gi-coupled mGluRs coexpressed with these G alpha subunits constitutes an easy functional assay for the pharmacological characterization of these receptors. The rank order of potency of antagonists was found to be (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine approximately (R,S)- alpha-methyl-4-phosphonophenylglycine > (R,S)-alpha-methyl-4-sulfonophenylglycine > (R,S)-alpha-methyl-4-tetrazolylphenylglycine = (S)-2-amino-2-methyl-4-phosphonobutyrate for mGluR2 and to be (R,S)-alpha-methyl-4-phosphonophenylglycine > or = (S)-2-amino-2-methyl-4-phosphonobutyrate > > (R,S)-alpha-methyl-4-sulfonophenylglycine [(R,S)-alpha-methyl-4-tetrazolylphenylglycine and (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine being inactive at 1 mM] for mGluR4. Using this functional assay, (R,S)-alpha-methyl-4-phosphonophenylglycine was found to have a similar KB value for mGluR2 and mGluR4.

    Molecular pharmacology 1996;50;4;923-30

  • Specificity of G alpha q and G alpha 11 gene expression in platelets and erythrocytes. Expressions of cellular differentiation and species differences.

    Johnson GJ, Leis LA and Dunlop PC

    Department of Medicine, VA Medical Center, Minneapolis, MN, USA.

    G alpha q and G alpha 11, members of the Gq family of G-proteins, transduce signals from receptors to the beta isoenzymes of phosphatidyl-inositol-specific phospholipase C (PI-PLC). The receptor specificity of these alpha subunits is unknown. G alpha q and G alpha 11 are ubiquitously expressed in tissues; however, there have been conflicting reports of the presence or absence of G alpha 11 protein in haematopoietic cells. Platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors activate PI-PLC via G alpha q, but the role of G alpha 11 is uncertain. To define their roles in platelet activation we studied G alpha q and G alpha 11 gene expression by immunotransfer blotting and by reverse transcription of mRNA followed by PCR (RT-PCR) and direct sequencing. An antiserum specific for mouse G alpha 11 failed to identify G alpha 11 in dog or human platelets or in dog liver, a tissue known to contain G alpha 11. RT-PCR performed with gene-specific primers demonstrated G alpha q mRNA, but not G alpha 11 mRNA, in normal human and mouse platelets and in thromboxane-sensitive and thromboxane-insensitive dog platelets. Studies of mouse and dog liver and human retina confirmed that the cDNA, primers and probes used could amplify and recognize G alpha 11 in other tissues. However, species-specific oligonucleotide primers and probes were essential to demonstrate G alpha 11, but not G alpha q, mRNA. Compared with mouse cDNA, dog and human G alpha 11 cDNA had twice as many nucleotide substitutions (approx. 12% compared with approx. 6%) as G alpha q, G alpha q mRNA was also found in mature erythrocytes but G alpha 11 mRNA was not identified, whereas both G alpha q and G alpha 11 mRNAs were found in bone marrow stem cells. Therefore G alpha 11 gene expression in haematopoietic cells is linked with cellular differentiation. The lack of G alpha 11 indicates that signal transduction from platelet TXA2/PGH2 receptors to PI-PLC occurs via G alpha q, and that G alpha 11 deficiency is not responsible for defective activation of PI-PLC in thromboxane-insensitive dog platelets. Despite the high degree of similarity that exists between G alpha q and G alpha 11, significantly greater species-specific variation in nucleotide sequence is present in G alpha 11 than in G alpha q. Cellular specificity and species specificity are important characteristics of these Gq family G-proteins.

    The Biochemical journal 1996;318 ( Pt 3);1023-31

  • Human G(alpha q): cDNA and tissue distribution.

    Chen B, Leverette RD, Schwinn DA and Kwatra MM

    Department of Anesthesiology and Pharmacology, Duke University Medical Center, Durham, North Carolina 27710, USA.

    G(alpha q), a member of the Gq family of heterotrimeric G proteins, transduces signals from several G protein-coupled receptors that stimulate membrane phosphoinositide hydrolysis. In order to further define the role of G(alpha q) in the function of G protein-coupled receptors, we have cloned the cDNA encoding human G(alpha q) from a prostate cDNA library. Human G(alpha q) exhibits high homology with its mouse homolog - 94% similarity at the nucleotide level, and 99% similarity at the amino acid level. Northern hybridization data indicate high expression of G(alpha q) mRNA in organs of the human reproductive system including ovary, prostate, and testis.

    Biochimica et biophysica acta 1996;1281;2;125-8

  • Differential distribution of alpha subunits and beta gamma subunits of heterotrimeric G proteins on Golgi membranes of the exocrine pancreas.

    Denker SP, McCaffery JM, Palade GE, Insel PA and Farquhar MG

    Division of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, 92093 USA.

    Heterotrimeric G proteins are well known to be involved in signaling via plasma membrane (PM) receptors. Recent data indicate that heterotrimeric G proteins are also present on intracellular membranes and may regulate vesicular transport along the exocytic pathway. We have used subcellular fractionation and immunocytochemical localization to investigate the distribution of G alpha and G beta gamma subunits in the rat exocrine pancreas which is highly specialized for protein secretion. We show that G alpha s, G alpha i3 and G alpha q/11 are present in Golgi fractions which are > 95% devoid of PM. Removal of residual PM by absorption on wheat germ agglutinin (WGA) did not deplete G alpha subunits. G alpha s was largely restricted to TGN-enriched fractions by immunoblotting, whereas G alpha i3 and G alpha q/11 were broadly distributed across Golgi fractions. G alpha s did not colocalize with TGN38 or caveolin, suggesting that G alpha s is associated with a distinct population of membranes. G beta subunits were barely detectable in purified Golgi fractions. By immunofluorescence and immunogold labeling, G beta subunits were detected on PM but not on Golgi membranes, whereas G alpha s and G alpha i3 were readily detected on both Golgi and PM. G alpha and G beta subunits were not found on membranes of zymogen granules. These data indicate that G alpha s, G alpha q/11, and G alpha i3 associate with Golgi membranes independent of G beta subunits and have distinctive distributions within the Golgi stack. G beta subunits are thought to lock G alpha in the GDP-bound form, prevent it from activating its effector, and assist in anchoring it to the PM. Therefore the presence of free G alpha subunits on Golgi membranes has several important functional implications: it suggests that G alpha subunits associated with Golgi membranes are in the active, GTP-bound form or are bound to some other unidentified protein(s) which can substitute for G beta gamma subunits. It further implies that G alpha subunits are tethered to Golgi membranes by posttranslational modifications (e.g., palmitoylation) or by binding to another protein(s).

    Funded by: NCI NIH HHS: CA58689; NIDDK NIH HHS: DK17780; NIGMS NIH HHS: GM40781

    The Journal of cell biology 1996;133;5;1027-40

  • The human thyrotropin receptor: a heptahelical receptor capable of stimulating members of all four G protein families.

    Laugwitz KL, Allgeier A, Offermanns S, Spicher K, Van Sande J, Dumont JE and Schultz G

    Institut für Pharmakologie, Freie Universität Berlin, Germany.

    Thyrotropin is the primary hormone that, via one heptahelical receptor, regulates thyroid cell functions such as secretion, specific gene expression, and growth. In human thyroid, thyrotropin receptor activation leads to stimulation of the adenylyl cyclase and phospholipase C cascades. However, the G proteins involved in thyrotropin receptor action have been only partially defined. In membranes of human thyroid gland, we immunologically identified alpha subunits of the G proteins Gs short, Gs long, Gi1, Gi2, Gi3, G(o) (Go2 and another form of Go, presumably Go1), Gq, G11, G12, and G13. Activation of the thyrotropin (TSH) receptor by bovine TSH led to increased incorporation of the photoreactive GTP analogue [alpha-32P]GTP azidoanilide into immunoprecipitated alpha subunits of all G proteins detected in thyroid membranes. This effect was receptor-dependent and not due to direct G protein stimulation because it was mimicked by TSH receptor-stimulating antibodies of patients suffering from Grave disease and was abolished by a receptor-blocking antiserum from a patient with autoimmune hypothyroidism. The TSH-induced activation of individual G proteins occurred with EC50 values of 5-50 milliunits/ml, indicating that the activated TSH receptor coupled with similar potency to different G proteins. When human thyroid slices were pretreated with pertussis toxin, the TSH receptor-mediated accumulation of cAMP increased by approximately 35% with TSH at 1 milliunits/ml, indicating that the TSH receptor coupled to Gs and G(i). Taken together, these findings show that, at least in human thyroid membranes, in which the protein is expressed at its physiological levels, the TSH receptor resembles a naturally occurring example of a general G protein-activating receptor.

    Proceedings of the National Academy of Sciences of the United States of America 1996;93;1;116-20

  • Molecular cloning of human G alpha q cDNA and chromosomal localization of the G alpha q gene (GNAQ) and a processed pseudogene.

    Dong Q, Shenker A, Way J, Haddad BR, Lin K, Hughes MR, McBride OW, Spiegel AM and Battey J

    Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-1752, USA. qhd@helix.nih.gov

    G alpha q is the alpha subunit of one of the heterotrimeric GTP-binding proteins that mediates stimulation of phospholipase C beta. We report the isolation and characterization of cDNA clones from a frontal cortex cDNA library encoding human G alpha q. The encoded protein is 359 amino acids long and is identical in all but one amino acid residue to mouse G alpha q. Analysis of human genomic DNA reveals an intronless sequence with strong homology to human G alpha q cDNA. In comparison to G alpha q cDNA, this genomic DNA sequence includes several small deletions and insertions that alter the reading frame, multiple single base changes, and a premature termination codon in the open reading frame, hallmarks of a processed pseudogene. Probes derived from human G alpha q cDNA sequence map to both chromosomes 2 and 9 in high-stringency genomic blot analyses of DNA from a panel of human-rodent hybrid cell lines. PCR primers that selectively amplify the pseudogene sequence generate a product only when DNA containing human chromosome 2 is used as the template, indicating that the authentic G alpha q gene (GNAQ) is located on chromosome 9. Regional localization by FISH analysis places GNAQ at 9q21 and the pseudogene at 2q14.3-q21.

    Genomics 1995;30;3;470-75

  • Ca2+ signalling in K562 human erythroleukaemia cells: effect of dimethyl sulphoxide and role of G-proteins in thrombin- and thromboxane A2-activated pathways.

    Thomas CP, Dunn MJ and Mattera R

    Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH, USA.

    The human leukaemic cell line K562 is a pluripotent stem cell with the potential to mature along a megakaryocytic or erythroid line. In these cells, thrombin and U46619 (9,11-dideoxy-9 alpha, 11 alpha-methanoepoxy prostaglandin F2 alpha), a thromboxane A2 analogue, increased intracellular Ca2+ in a rapid and concentration-dependent manner. The peak transient observed with both thrombin and U46619 was preserved upon stimulation in the absence of extracellular calcium and blunted with phorbol myristate acetate, suggestive of activation of phospholipase C. Short-term treatment with leupeptin abolished the calcium response to thrombin, but did not alter that to U46619. Both pertussis toxin (PT) and DMSO pretreatment inhibited thrombin- but not U46619-stimulated intracellular calcium elevation, indicating that these agonists signal through different G-proteins. Western blot analysis of crude membranes from K562 cells revealed the presence of G12 alpha and G13 alpha; the other known PT-substrates, Gi1 alpha and G0 alpha, were not detected. Consistent with this observation, ADP-ribosylation experiments revealed the presence of two PT substrates which co-migrated with human erythrocyte G12 alpha and G13 alpha. An antibody raised against Gq/11 alpha, a subfamily of G-protein alpha subunits unmodified by PT, specifically recognized 42 kDa protein(s) in K562 cells. PCR amplification of reverse-transcribed K562 RNA followed by DNA sequencing showed that these cells express messages for both Gq alpha and G11 alpha. Treatment of K562 cells with DMSO reduced the levels of thrombin receptor mRNA, without simultaneous changes in the expression of G12 alpha and G13 alpha. We have thus identified Ca(2+)-mobilizing agonists and related G-proteins in K562 cells, together with changes induced by DMSO in this signalling pathway.

    Funded by: NHLBI NIH HHS: HL-22563; NIGMS NIH HHS: GM 46552

    The Biochemical journal 1995;312 ( Pt 1);151-8

  • Mapping of single amino acid residues required for selective activation of Gq/11 by the m3 muscarinic acetylcholine receptor.

    Blin N, Yun J and Wess J

    Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.

    Each G protein-coupled receptor can interact only with a limited number of the many structurally similar G proteins expressed within a cell. This study was undertaken to identify single amino acids required for selectively coupling the m3 muscarinic acetylcholine receptor to G proteins of the Gq/11 family. To this goal, distinct intracellular segments/amino acids of the m3 receptor were systematically substituted into the structurally closely related m2 muscarinic receptor, which couples to Gi/o proteins, not Gq/11 proteins. The resultant mutant receptors were expressed in COS-7 cells and studied for their ability to induce agonist-dependent stimulation of phosphatidylinositol hydrolysis, a response known to be mediated by G proteins of the Gq/11 class. Using this approach, we were able to identify four amino acids in the second intracellular loop and four amino acids at the C terminus of the third intracellular loop of the m3 muscarinic receptor that are essential for efficient Gq/11 activation. We could demonstrate that these amino acids, together with a short segment at the N terminus of the third intracellular loop, fully account for the G protein coupling preference of the m3 muscarinic receptor. Taken together, our data strongly suggest that only a limited number of amino acids, located on different intracellular regions, are required to determine the functional profile of a given G protein-coupled receptor.

    The Journal of biological chemistry 1995;270;30;17741-8

  • Expression of GTP-binding protein alpha subunits in human thymocytes.

    Kabouridis PS, Waters ST, Escobar S, Stanners J and Tsoukas CD

    Department of Biology, San Diego State University, CA 92182, USA.

    In this report, we investigate G protein alpha subunit diversity in human thymocytes, utilizing common properties shared by these genes and reverse transcription-polymerase chain reaction (RT-PCR). Sequence analysis of PCR amplified gene portions, indicate the presence of members from all four G-protein families that have been described thus far. The alpha subunit genes identified are: G alpha i1-3 and G alpha z but not G alpha o from the Gi family, G alpha s from the Gs family, G alpha 11, G alpha q, and G alpha 16 from the Gq family, and G alpha 12 and G alpha 13 from the G12 family. Also in this report we present the nucleotide and predicted amino acid sequences of the human G alpha 13 cloned from a thymocyte cDNA library. The sequence of the human G alpha 13 has not been previously reported. Comparison of this sequence with the reported murine G alpha 13 shows > 90% identity at the deduced amino acid sequence level. We conclude that thymocytes represent a useful experimental system for the study of G protein involvement in immune responses and lymphocyte development.

    Funded by: NIGMS NIH HHS: GM39518, GM45765

    Molecular and cellular biochemistry 1995;144;1;45-51

  • Mechanism of GTP hydrolysis by G-protein alpha subunits.

    Kleuss C, Raw AS, Lee E, Sprang SR and Gilman AG

    Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235.

    Hydrolysis of GTP by a variety of guanine nucleotide-binding proteins is a crucial step for regulation of these biological switches. Mutations that impair the GTPase activity of certain heterotrimeric signal-transducing G proteins or of p21ras cause tumors in man. A conserved glutamic residue in the alpha subunit of G proteins has been hypothesized to serve as a general base, thereby activating a water molecule for nucleophilic attack on GTP. The results of mutagenesis of this residue (Glu-207) in Gi alpha 1 refute this hypothesis. Based on the structure of the complex of Gi alpha 1 with GDP, Mg2+, and AlF-4, which appears to resemble the transition state for GTP hydrolysis, we believe that Gln-204 of Gi alpha 1, rather than Glu-207, supports catalysis of GTP hydrolysis by stabilization of the transition state.

    Funded by: NIGMS NIH HHS: GM34497

    Proceedings of the National Academy of Sciences of the United States of America 1994;91;21;9828-31

  • The human thyrotropin receptor activates G-proteins Gs and Gq/11.

    Allgeier A, Offermanns S, Van Sande J, Spicher K, Schultz G and Dumont JE

    Institute of Interdisciplinary Research, University of Brussels, Belgium.

    The human thyrotropin receptor leads upon activation to the stimulation of phospholipase C and adenylyl cyclase. It is presently not known whether this bifurcating signaling occurs via two different G-proteins (Gq/11 and Gs) or via one G-protein (Gs). Receptor-activated Gs releases beta gamma subunits and alpha s, which then could regulate phospholipase C and adenylyl cyclase, respectively. In order to elucidate the signaling pathways induced by the activated thyroid-stimulating hormone (TSH) receptor, we studied the coupling of the TSH receptor to Gs and Gq/11 in human thyroid membranes. TSH concentration dependently led to the activation of two forms of Gs (Gs short and Gs long) as well as of Gq and G11, demonstrating that signaling pathways induced by TSH already bifurcate in the course of the receptor-G-protein interaction. These data strongly suggest the concept that phospholipase C and adenylyl cyclase activation through the TSH receptor are mediated by Gq/11 and Gs, respectively.

    The Journal of biological chemistry 1994;269;19;13733-5

  • Palmitoylation is required for signaling functions and membrane attachment of Gq alpha and Gs alpha.

    Wedegaertner PB, Chu DH, Wilson PT, Levis MJ and Bourne HR

    Department of Pharmacology, University of California, San Francisco 94143.

    We have identified the palmitoylated cysteine residues of alpha q and alpha s, alpha subunits of two heterotrimeric G proteins. Mutational substitutions of serines for cysteines 9 and 10 in alpha q and cysteine 3 in alpha s profoundly alter behavior of the subunits expressed in HEK293 cells. Neither mutant alpha subunit incorporates palmitate; both mutant proteins are found in the soluble rather than the particulate fraction; mutant alpha q or alpha s cannot couple a co-expressed receptor to stimulation of phospholipase C or adenylylcyclase, respectively; cysteine substitution prevents a mutationally activated alpha q (R183C) from stimulating phospholipase C directly, and reduces but does not abolish the ability of a similarly activated alpha s (R201C) to stimulate cAMP synthesis. Substitution of a myristoylation sequence for the palmitoylation sites leads to labeling of alpha q and alpha s by myristate, rather than by palmitate. Myristoylation restores the abilities of both nonpalmitoylated alpha q and alpha s to attach to membranes and, in the case of alpha q, restores its ability to stimulate phospholipase C, whether triggered by the R183C mutation or by receptor activation. These findings identify palmitoylation as a critical determinant of membrane attachment for alpha q and alpha s and show that this modification is required for normal signaling by these proteins.

    Funded by: NCI NIH HHS: CA-54427; NIGMS NIH HHS: GM-27800; NIMH NIH HHS: K21MH00961

    The Journal of biological chemistry 1993;268;33;25001-8

  • Identification and expression of G-proteins in human myometrium: up-regulation of G alpha s in pregnancy.

    Europe-Finner GN, Phaneuf S, Watson SP and López Bernal A

    University of Oxford, Nuffield Department of Obstetrics and Gynecology, John Radcliffe Hospital, Headington, United Kingdom.

    We report that human myometrium contains G alpha i1, G alpha i3, and G alpha q, and G alpha 11, which are expressed at similar levels in tissues from pregnant and nonpregnant women. G alpha i2 is also expressed, but at a slightly reduced level, in tissue taken from pregnant compared to nonpregnant donors. The major finding of this investigation is the substantial increase in G alpha s expression in pregnant myometrium. The increase in G alpha s levels may play a crucial role in maintaining relaxation of the uterus by favoring cAMP formation during pregnancy.

    Endocrinology 1993;132;6;2484-90

  • Signal-transducing G proteins and antidepressant drugs: evidence for modulation of alpha subunit gene expression in rat brain.

    Lesch KP and Manji HK

    Section of Neuropharmacology, National Institute of Mental Health, Bethesda, Maryland.

    Signal-transducing G proteins, heterotrimers formed of alpha, beta, and gamma subunits, are central to the coordination of receptor-effector communication. They are derived from a large gene family, and recent cloning and sequencing of cDNAs encoding the alpha subunits, which confer receptor and effector specificity on the heterotrimer, have defined four major classes, Gs, Gi, Gq, and G12, with at least 16 isotypes. The G proteins that coordinate receptor-effector activity are especially important in the central nervous system (CNS), where they serve widespread, critical roles in the regulation of neuronal function, maintain the functional balance between neurotransmitter systems, and, as such, represent attractive potential targets for antidepressant drugs. We describe an integrated series of animal and cell culture studies aimed at testing the hypothesis that alterations in G protein function may contribute the complex neuroadaptive mechanisms involved in the clinical actions of antidepressants, and demonstrate that long-term administration of a wide spectrum of antidepressant drugs regulate G alpha s, G alpha i1, G alpha i2, G alpha o, G alpha q, and G alpha 12 mRNA and protein expression in various areas of the rat brain. Additionally, we present the polymerase chain reaction-(PCR) mediated cross-species partial cDNA cloning and sequencing of rat and human G alpha o and rat G alpha 12, illustrate the regional distribution of G alpha mRNA and protein in rat brain, and provide evidence that different classes of antidepressants alter expression and/or stability of the recently identified G alpha 12 mRNA. We conclude that long-term treatment with antidepressant drugs exerts differential effects on G alpha mRNA and protein expression in rat brain, thus modifying signal transduction as an integral part of complex neuroadaptive mechanisms that may underlie their therapeutic efficacy. The development of novel drugs with G proteins as primary targets remains an attractive prospect for the future.

    Biological psychiatry 1992;32;7;549-79

  • Phospholipase C-beta 1 is a GTPase-activating protein for Gq/11, its physiologic regulator.

    Berstein G, Blank JL, Jhon DY, Exton JH, Rhee SG and Ross EM

    Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235-9041.

    Purified M1 muscarinic cholinergic receptor and Gq/11 were coreconstituted in lipid vesicles. Addition of purified phospholipase C-beta 1 (PLC-beta 1) further stimulated the receptor-promoted steady-state GTPase activity of Gq/11 up to 20-fold. Stimulation depended upon receptor-mediated GTP-GDP exchange. Addition of PLC-beta 1 caused a rapid burst of hydrolysis of Gq/11-bound GTP that was at least 50-fold faster than in its absence. Thus, PLC-beta 1 stimulates hydrolysis of Gq/11-bound GTP and acts as a GTPase-activating protein (GAP) for its physiologic regulator, Gq/11. GTPase-stimulating activity was specific both for PLC-beta 1 and Gq/11. Such GAP activity by an effector coupled to a trimeric G protein can reconcile slow GTP hydrolysis by pure G proteins in vitro with fast physiologic deactivation of G protein-mediated signaling.

    Funded by: FIC NIH HHS: TW04475; NIGMS NIH HHS: GM30355

    Cell 1992;70;3;411-8

  • The G protein coupled to the thromboxane A2 receptor in human platelets is a member of the novel Gq family.

    Shenker A, Goldsmith P, Unson CG and Spiegel AM

    Molecular Pathophysiology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.

    The thromboxane A2 (TXA2) receptor in human platelets is coupled to a pertussis toxin-insensitive G protein whose identity has remained unknown. Candidates for this role include the atypical G protein known as Gz and members of a recently discovered G protein family known as Gq. Because of the proven utility of antibodies directed against the C terminus of G protein alpha subunits as functional probes, we prepared an antibody against a synthetic decapeptide corresponding to the C-terminal sequence shared by alpha 11 and alpha q, two members of the new family. This antibody (QL) does not recognize known alpha subunits but selectively binds to a 42-kDa protein in a variety of tissues, including human platelet membranes. QL and two other C-terminal antibodies, QN and AS, known to recognize alpha z and alpha i2, respectively, were tested for their ability to block agonist-stimulated GTPase activity in human platelet membranes. Pretreatment of platelet membranes with AS has previously been shown to interfere with alpha 2 adrenergic receptor-mediated inhibition of adenylylcyclase. As expected, only AS antibody produced inhibition of alpha 2 receptor-stimulated GTPase. Pretreatment of membranes with QL, but not QN or AS, caused marked inhibition of TXA2 receptor-stimulated GTPase. This identifies the G protein coupled to human platelet TXA2 receptors as a member of the novel Gq family.

    The Journal of biological chemistry 1991;266;14;9309-13

  • Purification and characterization of membrane-bound phospholipase C specific for phosphoinositides from human platelets.

    Banno Y, Yada Y and Nozawa Y

    Department of Biochemistry, Gifu University School of Medicine, Japan.

    Two peaks (mPLC-I and mPLC-II) of phosphatidylinositol 4,5-bisphosphate (PIP2)-hydrolyzing activity were resolved when 1% sodium cholate extract from particulate fractions of human platelet was chromatographed on a heparin-Sepharose column. The major peak of enzyme activity (mPLC-II) was purified to homogeneity by a combination of Fast Q-Sepharose, heparin-Sepharose, Ultrogel AcA-44, Mono Q, Superose 6-12 combination column, and Superose 12 column chromatographies. The specific activity increased 2,700-fold as compared with that of the starting particulate fraction. The purified mPLC-II had an estimated molecular weight of 61,000 on sodium dodecyl sulfate-polyacrylamide gels. The minor peak of enzyme activity (mPLC-I) was partially purified to 430-fold. Both enzymes hydrolyzed PIP2 at low Ca2+ concentration (0.1-10 microM) and exhibited higher Vmax for PIP2 than for phosphatidylinositol. PIP2-hydrolyzing activities of both enzymes were enhanced by various detergents and lipids, such as deoxycholate, cholate, phosphatidylethanolamine, and dimyristoylphosphatidylcholine. The mPLC-I and mPLC-II activities were increased by Ca2+, but not by Mg2+, while Hg2+, Fe2+, Cu2+, and La3+ were inhibitory. GTP-binding proteins (Gi, Go, and Ki-ras protein) had no significant effects on the mPLC-II activity.

    The Journal of biological chemistry 1988;263;23;11459-65

Gene lists (5)

Gene List Source Species Name Description Gene count
L00000013 G2C Homo sapiens Human mGluR5 Human orthologues of mouse mGluR5 complex adapted from Collins et al (2006) 52
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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