G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
adrenergic, beta, receptor kinase 1
G00000157 (Mus musculus)

Databases (7)

ENSG00000173020 (Ensembl human gene)
156 (Entrez Gene)
507 (G2Cdb plasticity & disease)
ADRBK1 (GeneCards)
109635 (OMIM)
Marker Symbol
Protein Sequence
P25098 (UniProt)

Synonyms (2)

  • BARK1
  • GRK2

Literature (108)

Pubmed - other

  • Role of helix 8 of the thyrotropin-releasing hormone receptor in phosphorylation by G protein-coupled receptor kinase.

    Gehret AU, Jones BW, Tran PN, Cook LB, Greuber EK and Hinkle PM

    Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, NY 14642, USA.

    The thyrotropin-releasing hormone (TRH) receptor undergoes rapid and extensive agonist-dependent phosphorylation attributable to G protein-coupled receptor (GPCR) kinases (GRKs), particularly GRK2. Like many GPCRs, the TRH receptor is predicted to form an amphipathic helix, helix 8, between the NPXXY motif at the cytoplasmic end of the seventh transmembrane domain and palmitoylation sites at Cys335 and Cys337. Mutation of all six lysine and arginine residues between the NPXXY and residue 340 to glutamine (6Q receptor) did not prevent the receptor from stimulating inositol phosphate turnover but almost completely prevented receptor phosphorylation in response to TRH. Phosphorylation at all sites in the cytoplasmic tail was inhibited. The phosphorylation defect was not reversed by long incubation times or high TRH concentrations. As expected for a phosphorylation-defective receptor, the 6Q-TRH receptor did not recruit arrestin, undergo the typical arrestin-dependent increase in agonist affinity, or internalize well. Lys326, directly before phenylalanine in the common GPCR motif NPXXY(X)(5-6)F(R/K), was critical for phosphorylation. The 6Q-TRH receptor was not phosphorylated effectively in cells overexpressing GRK2 or in in vitro kinase assays containing purified GRK2. Phosphorylation of the 6Q receptor was partially restored by coexpression of a receptor with an intact helix 8 but without phosphorylation sites. Phosphorylation was inhibited but not completely prevented by alanine substitution for cysteine palmitoylation sites. Positively charged amino acids in the proximal tail of the beta2-adrenergic receptor were also important for GRK-dependent phosphorylation. The results indicate that positive residues in helix 8 of GPCRs are important for GRK-dependent phosphorylation.

    Funded by: NIDDK NIH HHS: DK19974, R01 DK019974-25, R01 DK019974-33

    Molecular pharmacology 2010;77;2;288-97

  • G protein-coupled receptor kinase-2 constitutively regulates D2 dopamine receptor expression and signaling independently of receptor phosphorylation.

    Namkung Y, Dipace C, Urizar E, Javitch JA and Sibley DR

    Molecular Neuropharmacology Section, NINDS, National Institutes of Health, Bethesda, Maryland 20892-9405, USA.

    We investigated the regulatory effects of GRK2 on D(2) dopamine receptor signaling and found that this kinase inhibits both receptor expression and functional signaling in a phosphorylation-independent manner, apparently through different mechanisms. Overexpression of GRK2 was found to suppress receptor expression at the cell surface and enhance agonist-induced internalization, whereas short interfering RNA knockdown of endogenous GRK2 led to an increase in cell surface receptor expression and decreased agonist-mediated endocytosis. These effects were not due to GRK2-mediated phosphorylation of the D(2) receptor as a phosphorylation-null receptor mutant was regulated similarly, and overexpression of a catalytically inactive mutant of GRK2 produced the same effects. The suppression of receptor expression is correlated with constitutive association of GRK2 with the receptor complex as we found that GRK2 and several of its mutants were able to co-immunoprecipitate with the D(2) receptor. Agonist pretreatment did not enhance the ability of GRK2 to co-immunoprecipitate with the receptor. We also found that overexpression of GRK2 attenuated the functional coupling of the D(2) receptor and that this activity required the kinase activity of GRK2 but did not involve receptor phosphorylation, thus suggesting the involvement of an additional GRK2 substrate. Interestingly, we found that the suppression of functional signaling also required the G betagamma binding activity of GRK2 but did not involve the GRK2 N-terminal RH domain. Our results suggest a novel mechanism by which GRK2 negatively regulates G protein-coupled receptor signaling in a manner that is independent of receptor phosphorylation.

    Funded by: Intramural NIH HHS; NIDA NIH HHS: DA022413, K05 DA022413; NIMH NIH HHS: MH54137, R01 MH054137

    The Journal of biological chemistry 2009;284;49;34103-15

  • The G protein coupled receptor kinase 2 plays an essential role in beta-adrenergic receptor-induced insulin resistance.

    Cipolletta E, Campanile A, Santulli G, Sanzari E, Leosco D, Campiglia P, Trimarco B and Iaccarino G

    Dipartimento di Medicina Clinica, Medicina Clinica, Scienze Cardiovascolari ed Immunologiche, Università Federico II, Via Pansini 5, 80131 Naples, Italy.

    Aims: Insulin (Ins) resistance (IRES) associates to increased cardiovascular risk as observed in metabolic syndrome. Chronic stimulation of beta-adrenergic receptors (betaAR) due to exaggerated sympathetic nervous system activity is involved in the pathogenesis of IRES. The cellular levels of G protein coupled receptor kinase 2 (GRK2) increase during chronic betaAR stimulation, leading to betaAR desensitization. We tested the hypothesis that GRK2 plays a role in betaAR-induced IRES.

    We evaluated Ins-induced glucose uptake and signalling responses in vitro in cell overexpressing the beta(2)AR, the GRK2, or the catalytically dead mutant GRK2-DN. In a model of increased adrenergic activity, IRES and elevated cellular GRK2 levels, the spontaneously hypertensive rats (SHR) we performed the intravenous glucose tolerance test load. To inhibit GRK2, we synthesized a peptide based on the catalytical sequence of GRK2 conjugated with the antennapedia internalization sequence (Ant-124). Ins in human kidney embryonic (HEK-293) cells causes rapid accumulation of GRK2, tyrosine phosphorylation of Ins receptor substrate 1 (IRS1) and induces glucose uptake. In the same cell type, transgenic beta(2)AR overexpression causes GRK2 accumulation associated with significant deficit of IRS1 activation and glucose uptake by Ins. Similarly, transgenic GRK2 overexpression prevents Ins-induced tyrosine phosphorylation of IRS1 and glucose uptake, whereas GRK2-DN ameliorates glucose extraction. By immunoprecipitation, GRK2 binds IRS1 but not the Ins receptor in an Ins-dependent fashion, which is lost in HEK-GRK2 cells. Ant-124 improves Ins-induced glucose uptake in HEK-293 and HEK-GRK2 cells, but does not prevent GRK2/IRS1 interaction. In SHR, Ant-124 infusion for 30 days ameliorates IRES and IRS1 tyrosine phosphorylation.

    Conclusion: Our results suggest that GRK2 mediates adrenergic IRES and that inhibition of GRK2 activity leads to increased Ins sensitivity both in cells and in animal model of IRES.

    Cardiovascular research 2009;84;3;407-15

  • Regulation of GPR54 signaling by GRK2 and {beta}-arrestin.

    Pampillo M, Camuso N, Taylor JE, Szereszewski JM, Ahow MR, Zajac M, Millar RP, Bhattacharya M and Babwah AV

    Children's Health Research Institute, Department of Obstetrics and Gynaecology, The University of Western Ontario, London, Canada.

    Kisspeptin and its receptor, GPR54, are major regulators of the hypothalamic-pituitary-gonadal axis as well as regulators of human placentation and tumor metastases. GPR54 is a G(q/11)-coupled G protein-coupled receptor (GPCR), and activation by kisspeptin stimulates phosphatidy linositol 4, 5-biphosphate hydrolysis, Ca(2+) mobilization, arachidonic acid release, and ERK1/2 MAPK phosphorylation. Physiological evidence suggests that GPR54 undergoes agonist-dependent desensitization, but underlying molecular mechanisms are unknown. Furthermore, very little has been reported on the early events that regulate GPR54 signaling. The lack of information in these important areas led to this study. Here we report for the first time on the role of GPCR serine/threonine kinase (GRK)2 and beta-arrestin in regulating GPR54 signaling in human embryonic kidney (HEK) 293 cells, a model cell system for studying the molecular regulation of GPCRs, and genetically modified MDA MB-231 cells, an invasive breast cancer cell line expressing about 75% less beta-arrestin-2 than the control cell line. Our study reveals that in HEK 293 cells, GPR54 is expressed both at the plasma membrane and intracellularly and also that plasma membrane expression is regulated by cytoplasmic tail sequences. We also demonstrate that GPR54 exhibits constitutive activity, internalization, and association with GRK2 and beta- arrestins-1 and 2 through sequences in the second intracellular loop and cytoplasmic tail of the receptor. We also show that GRK2 stimulates the desensitization of GPR54 in HEK 293 cells and that beta-arrestin-2 mediates GPR54 activation of ERK1/2 in MDA-MB-231 cells. The significance of these findings in developing molecular-based therapies for treating certain endocrine-related disorders is discussed.

    Funded by: Canadian Institutes of Health Research: MOP 81383

    Molecular endocrinology (Baltimore, Md.) 2009;23;12;2060-74

  • Decreased GRK3 but not GRK2 expression in frontal cortex from bipolar disorder patients.

    Rao JS, Rapoport SI and Kim HW

    Brain Physiology and Metabolism Section, NIA, NIH, Bethesda, MD 20892, USA. jrao@grc.nia.nih.gov

    Overactivation of G-protein-mediated functions and altered G-protein regulation have been reported in bipolar disorder (BD) brain. Further, drugs effective in treating BD are reported to up-regulate expression of G-protein receptor kinase (GRK) 3 in rat frontal cortex. We therefore hypothesized that some G-protein subunits and GRK levels would be reduced in the brain of BD patients. We determined protein and mRNA levels of G-protein beta and gamma subunits, GRK2, and GRK3 in post-mortem frontal cortex from 10 BD patients and 10 age-matched controls by using immunoblots and real-time RT-PCR. There were statistically significant decreases in protein and mRNA levels of G-protein subunits beta and gamma and of GRK3 in BD brain but not a significant difference in the GRK2 level. Decreased expression of G-protein subunits and of GRK3 may alter neurotransmission, leading to disturbed cognition and behaviour in BD.

    Funded by: NIA NIH HHS: Z01 AG000145-07

    The international journal of neuropsychopharmacology / official scientific journal of the Collegium Internationale Neuropsychopharmacologicum (CINP) 2009;12;6;851-60

  • Dual regulation of lysophosphatidic acid (LPA1) receptor signalling by Ral and GRK.

    Aziziyeh AI, Li TT, Pape C, Pampillo M, Chidiac P, Possmayer F, Babwah AV and Bhattacharya M

    Department of Physiology and Pharmacology, The University of Western Ontario, London, Ontario, Canada.

    Lysophosphatidic acid (LPA) is a major constituent of blood and is involved in a variety of physiological and pathophysiological processes. LPA signals via the ubiquitously expressed G protein-coupled receptors (GPCRs), LPA(1) and LPA(2) that are specific for LPA. However, in large, the molecular mechanisms that regulate the signalling of these receptors are unknown. We show that the small GTPase RalA associates with both LPA(1) and LPA(2) in human embryonic kidney (HEK 293) cells and that stimulation of LPA(1) receptors with LPA triggers the activation of RalA. While RalA was not found to play a role in the endocytosis of LPA receptors, we reveal that LPA(1) receptor stimulation promoted Ral-dependent phospholipase C activity. Furthermore, we found that GRK2 is required for the desensitization of LPA(1) and LPA(2) and have identified a novel interaction between RalA and GRK2, which is promoted by LPA(1) receptor activity. Taken together, these results establish RalA and GRK2 as key regulators of LPA receptor signalling and demonstrate for the first time that LPA(1) activity facilitates the formation of a novel protein complex between these two proteins.

    Cellular signalling 2009;21;7;1207-17

  • G protein-coupled receptor kinase 2 expression and activity are associated with blood pressure in black Americans.

    Cohn HI, Xi Y, Pesant S, Harris DM, Hyslop T, Falkner B and Eckhart AD

    Center for Translational Medicine, Thomas Jefferson Hospital, Philadelphia, PA, USA.

    Hypertension occurs with higher prevalence and morbidity in black Americans compared with other groups. Alterations in the signal transduction pathways of 7-transmembrane spanning receptors are found in hypertensive patients. G protein-coupled receptor kinases (GRKs) play an important role in regulating this receptor signaling. The 2 most abundantly expressed GRKs in the cardiovascular system are GRK2 and GRK5, and each has unique substrates. Understanding changes in expression may give us insight into activated receptors in the pathophysiological progression of hypertension. In heart failure and white hypertensives, increased GRK2 expression arises because of neurohormonal stimulation of particular receptors. GRK2 subsequently desensitizes specific receptors, including beta-adrenergic receptors. In blood pressure control, beta-adrenergic receptor desensitization could lead to increased blood pressure. GRK2 and GRK5 mRNA were evaluated in lymphocytes of black Americans via quantitative real-time PCR. GRK2 mRNA expression directly correlated with systolic blood pressure and norepinephrine levels. GRK2 was elevated >30% among those with systolic blood pressure > or =130 mm Hg. No significant correlation between GRK5 mRNA expression and blood pressure or catecholamines was observed. Diabetic status, age, sex, and body mass index were also compared with GRK2 expression using univariate and multivariate analyses. GRK2 protein expression was elevated 2-fold in subjects with higher blood pressure, and GRK activity was increased >40%. Our data suggest that GRK2, but not GRK5, is correlated with increasing blood pressure in black Americans. Understanding the receptors stimulated by increased neurohormonal activation may give insight into the pathophysiology of hypertension in this at-risk population.

    Funded by: NIDDK NIH HHS: R01 DK046107, R01 DK046107-08, R01 DK046107-09, R01 DK046107-10

    Hypertension (Dallas, Tex. : 1979) 2009;54;1;71-6

  • GRK2 activation by receptors: role of the kinase large lobe and carboxyl-terminal tail.

    Sterne-Marr R, Leahey PA, Bresee JE, Dickson HM, Ho W, Ragusa MJ, Donnelly RM, Amie SM, Krywy JA, Brookins-Danz ED, Orakwue SC, Carr MJ, Yoshino-Koh K, Li Q and Tesmer JJ

    Biology Department, Siena College, Loudonville, New York 12211, USA. sternemarr@siena.edu

    G protein-coupled receptor (GPCR) kinases (GRKs) were discovered by virtue of their ability to phosphorylate activated GPCRs. They constitute a branch of the AGC kinase superfamily, but their mechanism of activation is largely unknown. To initiate a study of GRK2 activation, we sought to identify sites on GRK2 remote from the active site that are involved in interactions with their substrate receptors. Using the atomic structure of GRK2 in complex with Gbetagamma as a guide, we predicted that residues on the surface of the kinase domain that face the cell membrane would interact with the intracellular loops and carboxyl-terminal tail of the GPCR. Our study focused on two regions: the kinase large lobe and an extension of the kinase domain known as the C-tail. Residues in the GRK2 large lobe whose side chains are solvent exposed and facing the membrane were targeted for mutagenesis. Residues in the C-tail of GRK2, although not ordered in the crystal structure, were also targeted because this region has been implicated in receptor binding and in the regulation of AGC kinase activity. Four substitutions out of 20, all within or adjacent to the C-tail, resulted in significant deficiencies in the ability of the enzyme to phosphorylate two different GPCRS: rhodopsin, and the beta(2)-adrenergic receptor. The mutant exhibiting the most dramatic impairment, V477D, also showed significant defects in phosphorylation of nonreceptor substrates. Interestingly, Michaelis-Menten kinetics suggested that V477D had a 12-fold lower k(cat), but no changes in K(M), suggesting a defect in acquisition or stabilization of the closed state of the kinase domain. V477D was also resistant to activation by agonist-treated beta(2)AR. Therefore, Val477 and other residues in the C-tail are expected to play a role in the activation of GRK2 by GPCRs.

    Funded by: NHLBI NIH HHS: HL071818, HL086865, R01 HL071818, R01 HL071818-04, R01 HL071818-05, R01 HL071818-06, R01 HL086865, R01 HL086865-01

    Biochemistry 2009;48;20;4285-93

  • Insights into cerebrovascular complications and Alzheimer disease through the selective loss of GRK2 regulation.

    Obrenovich ME, Morales LA, Cobb CJ, Shenk JC, Méndez GM, Fischbach K, Smith MA, Qasimov EK, Perry G and Aliev G

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

    Alzheimer disease (AD) and stroke are two leading causes of age-associated dementia. Increasing evidence points to vascular damage as an early contributor to the development of AD and AD-like pathology. In this review, we discuss the role of G protein-coupled receptor kinase 2 (GRK2) as it relates to individuals affected by AD and how the cardiovasculature plays a role in AD pathogenesis. The possible involvement of GRKs in AD pathogenesis is an interesting notion, which may help bridge the gap in our understanding of the heartbrain connection in relation to neurovisceral damage and vascular complications in AD, since kinases of this family are known to regulate numerous receptor functions both in the brain, myocardium, and elsewhere. The aim of this review is to discuss our findings of overexpression of GRK2 in the context of the early pathogenesis of AD, because increased levels of GRK2 immunoreactivity were found in vulnerable neurons of AD patients as well as in a two-vessel occlusion (2-VO) mammalian model of ischaemia. Also, we consider the consequences for this overexpression as a loss of G-protein coupled receptor (GPCR) regulation, as well as suggest a potential role for GPCRs and GRKs in a unifying theory of AD pathogenesis, particularly in the context of cerebrovascular disease. We synthesize this newer information and attempt to put it into context with GRKs as regulators of diverse physiological cellular functions that could be appropriate targets for future pharmacological intervention.

    Funded by: NIGMS NIH HHS: R25 GM060655-08

    Journal of cellular and molecular medicine 2009;13;5;853-65

  • [Genetics of atrial fibrillation].

    Nikulina SIu, Shul'man VA, Kuznetsova OO, Aksiutina NV, Chernova AA, Maksimov VN, Kulikov IV, Ustinov SN, Kazarinova IuL, Romashchenko AG and Voevoda MI

    We carried out examination of 103 probands with atrial fibrillation (AF) and 301 their 1st, 2nd, and 3rd degree relatives (main group). In addition we examined 82 probands without clinical electrocardiographic signs of heart disease and 163 their 1st and 2nd degree relatives (control group). We found accumulation of AF in families of probands with this pathology. Segregation analysis of idiopathic forms of AF allowed to reveal autosomal dominant type of inheritance of this pathology. Heterozygous variant of Ser49Gly of betai-adrenoreceptor gene can be considered as one of genetic predictors of development of how primary and secondary AF.

    Kardiologiia 2009;49;3;43-8

  • Selective regulation of H1 histamine receptor signaling by G protein-coupled receptor kinase 2 in uterine smooth muscle cells.

    Willets JM, Taylor AH, Shaw H, Konje JC and Challiss RA

    Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, University of Leicester, Clinical Sciences Building, Leicester Royal Infirmary, Leicester LE2 7LX, United Kingdom. jmw23@le.ac.uk

    Histamine stimulates uterine contraction; however, little is known regarding the mechanism or regulation of uterine histamine receptor signaling. Here we investigated the regulation of Galpha(q/11)-coupled histamine receptor signaling in human myometrial smooth muscle cells using the inositol 1,4,5-trisphosphate biosensor pleckstrin homology domain of phospholipase-delta1 tagged to enhanced green fluorescent protein and the Ca(2+)-sensitive dye Fluo-4. Histamine addition caused concentration-dependent increases in inositol 1,4,5-trisphosphate and [Ca(2+)](i) in the ULTR human uterine smooth muscle cell line and primary human myometrial cells. These effects were completely inhibited by the H(1) histamine receptor antagonist, diphenhydramine, and were unaffected by the H(2) histamine receptor antagonist, cimetidine. ULTR and primary myometrial cells were transfected with either dominant-negative G protein-coupled receptor kinases (GRKs) or small interfering RNAs targeting specific GRKs to assess the roles of this protein kinase family in H(1) histamine receptor desensitization. Dominant-negative GRK2, but not GRK5 or GRK6, prevented H(1) histamine receptor desensitization. Similarly, transfection with short interfering RNAs (that each caused >70% depletion of the targeted GRK) for GRK2, but not GRK3 or GRK6, also prevented H(1) histamine receptor desensitization. Our data suggest that histamine stimulates phospholipase C-signaling in myometrial smooth muscle cells through H(1) histamine receptors and that GRK2 recruitment is a key mechanism in the regulation of H(1) histamine receptor signaling in human uterine smooth muscle. These data provide insights into the in situ regulation of this receptor subtype and may inform pathophysiological functioning in preterm labor and other conditions involving uterine smooth muscle dysregulation.

    Molecular endocrinology (Baltimore, Md.) 2008;22;8;1893-907

  • EGF transregulates opioid receptors through EGFR-mediated GRK2 phosphorylation and activation.

    Chen Y, Long H, Wu Z, Jiang X and Ma L

    Pharmacology Research Center and State Key Laboratory of Medical Neurobiology, Shanghai Medical College and Institutes of Brain Science, Fudan University, Shanghai 200032, China.

    G protein-coupled receptor (GPCR) kinases (GRKs) are key regulators of GPCR function. Here we demonstrate that activation of epidermal growth factor receptor (EGFR), a member of receptor tyrosine kinase family, stimulates GRK2 activity and transregulates the function of G protein-coupled opioid receptors. Our data showed that EGF treatment promoted DOR internalization induced by DOR agonist and this required the intactness of GRK2-phosphorylation sites in DOR. EGF stimulation induced the association of GRK2 with the activated EGFR and the translocation of GRK2 to the plasma membrane. After EGF treatment, GRK2 was phosphorylated at tyrosyl residues. Mutational analysis indicated that EGFR-mediated phosphorylation occurred at GRK2 N-terminal tyrosyl residues previously shown as c-Src phosphorylation sites. However, c-Src activity was not required for EGFR-mediated phosphorylation of GRK2. In vitro assays indicated that GRK2 was a direct interactor and a substrate of EGFR. EGF treatment remarkably elevated DOR phosphorylation in cells expressing the wild-type GRK2 in an EGFR tyrosine kinase activity-dependent manner, whereas EGF-stimulated DOR phosphorylation was greatly decreased in cells expressing mutant GRK2 lacking EGFR tyrosine kinase sites. We further showed that EGF also stimulated internalization of mu-opioid receptor, and this effect was inhibited by GRK2 siRNA. These data indicate that EGF transregulates opioid receptors through EGFR-mediated tyrosyl phosphorylation and activation of GRK2 and propose GRK2 as a mediator of cross-talk from RTK to GPCR signaling pathway.

    Molecular biology of the cell 2008;19;7;2973-83

  • Immunohistochemical detection, regulation and antiproliferative function of G-protein-coupled receptor kinase 2 in thyroid carcinomas.

    Métayé T, Levillain P, Kraimps JL and Perdrisot R

    Biophysics Laboratory, CHU de Poitiers, BP 577, 86021 Poitiers Cedex, France. t.metaye@chu-poitiers.fr

    TSH, via its G-protein-coupled receptor, activates cell growth of both benign and malignant thyroid tumors. G-protein-coupled receptors (GR) kinase 2 (GRK2) has been reported to regulate the TSH receptor but its role in cancer is unknown. To determine a possible function for GRK2 in the growth process of thyroid cancers, we analysed its expression in normal and tumoral thyroid tissues and studied thyroid cancer cell line proliferation after GRK2 overexpression. Thirty one thyroid tissues, including 16 non-medullary thyroid cancers and 15 adjacent normal tissues, were analysed by immunohistochemistry. Five paired tissues were also studied by western blotting for the GRK2 enzymatic activity. Immunohistochemical staining showed an increase in GRK2 in thyroid cancers including papillary, follicular, and anaplastic types, compared with their adjacent normal tissues. Immunoblot analysis and GRK2 enzymatic activity measurement confirmed immunohistochemical study. TSH and TSH in association with insulin or IGF-I stimulated GRK2 protein accumulation in normal human thyroid cells in primary culture. The TSH effect on the GRK2 expression was mimicked by forskolin. After GRK2 overexpression in two poorly differentiated thyroid cell lines, all the clones showed a significant reduction in cell proliferation, ranging from 28 to 65% inhibition compared with vector alone after 96-h culture. In conclusion, thyroid mitogenic factor-stimulated GRK2 accumulation may explain, in part, high GRK2 levels in differentiated carcinoma, because TSH, insulin, or IGF-I is known to be involved in the thyroid cancer progression. Surprisingly, instead of stimulating, GRK2 reduced cell proliferation revealing a new role for this kinase in the growth of thyroid cancers.

    The Journal of endocrinology 2008;198;1;101-10

  • A GRK5 polymorphism that inhibits beta-adrenergic receptor signaling is protective in heart failure.

    Liggett SB, Cresci S, Kelly RJ, Syed FM, Matkovich SJ, Hahn HS, Diwan A, Martini JS, Sparks L, Parekh RR, Spertus JA, Koch WJ, Kardia SL and Dorn GW

    Department of Internal Medicine, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, Ohio 45267, USA.

    Beta-adrenergic receptor (betaAR) blockade is a standard therapy for cardiac failure and ischemia. G protein-coupled receptor kinases (GRKs) desensitize betaARs, suggesting that genetic GRK variants might modify outcomes in these syndromes. Re-sequencing of GRK2 and GRK5 revealed a nonsynonymous polymorphism of GRK5, common in African Americans, in which leucine is substituted for glutamine at position 41. GRK5-Leu41 uncoupled isoproterenol-stimulated responses more effectively than did GRK5-Gln41 in transfected cells and transgenic mice, and, like pharmacological betaAR blockade, GRK5-Leu41 protected against experimental catecholamine-induced cardiomyopathy. Human association studies showed a pharmacogenomic interaction between GRK5-Leu41 and beta-blocker treatment, in which the presence of the GRK5-Leu41 polymorphism was associated with decreased mortality in African Americans with heart failure or cardiac ischemia. In 375 prospectively followed African-American subjects with heart failure, GRK5-Leu41 protected against death or cardiac transplantation. Enhanced betaAR desensitization of excessive catecholamine signaling by GRK5-Leu41 provides a 'genetic beta-blockade' that improves survival in African Americans with heart failure, suggesting a reason for conflicting results of beta-blocker clinical trials in this population.

    Funded by: NHLBI NIH HHS: HL77113, P50 HL077101-03, P50 HL077101-039001, P50 HL077101-039003, P50 HL077101-040002, P50 HL77101, R01 HL087871-01, R01 HL087871-06, R01 HL87871

    Nature medicine 2008;14;5;510-7

  • G protein-coupled receptor kinase 2 positively regulates epithelial cell migration.

    Penela P, Ribas C, Aymerich I, Eijkelkamp N, Barreiro O, Heijnen CJ, Kavelaars A, Sánchez-Madrid F and Mayor F

    Departamento de Biología Molecular and Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Madrid, Spain. ppenela@cbm.uam.es

    Cell migration requires integration of signals arising from both the extracellular matrix and messengers acting through G protein-coupled receptors (GPCRs). We find that increased levels of G protein-coupled receptor kinase 2 (GRK2), a key player in GPCR regulation, potentiate migration of epithelial cells towards fibronectin, whereas such process is decreased in embryonic fibroblasts from hemizygous GRK2 mice or upon knockdown of GRK2 expression. Interestingly, the GRK2 effect on fibronectin-mediated cell migration involves the paracrine/autocrine activation of a sphingosine-1-phosphate (S1P) Gi-coupled GPCR. GRK2 positively modulates the activity of the Rac/PAK/MEK/ERK pathway in response to adhesion and S1P by a mechanism involving the phosphorylation-dependent, dynamic interaction of GRK2 with GIT1, a key scaffolding protein in cell migration processes. Furthermore, decreased GRK2 levels in hemizygous mice result in delayed wound healing rate in vivo, consistent with a physiological role of GRK2 as a regulator of coordinated integrin and GPCR-directed epithelial cell migration.

    The EMBO journal 2008;27;8;1206-18

  • Characterization of G-protein coupled receptor kinase interaction with the neurokinin-1 receptor using bioluminescence resonance energy transfer.

    Jorgensen R, Holliday ND, Hansen JL, Vrecl M, Heding A, Schwartz TW and Elling CE

    7TM Pharma A/S, 2970 Horsholm, Denmark.

    To analyze the interaction between the neurokinin-1 (NK-1) receptor and G-protein coupled receptor kinases (GRKs), we performed bioluminescence resonance energy transfer(2) (BRET(2)) measurements between the family A NK-1 receptor and GRK2 and GRK5 as well as their respective kinase-inactive mutants. We observed agonist induced interaction of both GRK5 and GRK2 with the activated NK-1 receptor. In saturation experiments, we observed GRK5 to interact with the activated receptor in a monophasic manner while GRK2 interacted in a biphasic manner with the low affinity phase corresponding to receptor affinity for GRK5. Agonist induced GRK5 interaction with the receptor was dependent on intact kinase-activity, whereas the high affinity phase of GRK2 interaction was independent of kinase activity. We were surprised to find that the BRET(2) saturation experiments indicated that before receptor activation, the full-length NK-1 receptor, but not a functional C-terminal tail-truncated receptor, is preassociated with GRK5 in a relatively low-affinity state. We demonstrate that GRK5 can compete for agonist induced GRK2 interaction with the NK-1 receptor, whereas GRK2 does not compete for receptor interaction with GRK5. We suggest that GRK5 is preassociated with the NK-1 receptor and that GRK5, rather than GRK2, is a key player in competitive regulation of GRK subtype specific interaction with the NK-1 receptor.

    Molecular pharmacology 2008;73;2;349-58

  • Dynamic regulation of phosphoinositide 3-kinase-gamma activity and beta-adrenergic receptor trafficking in end-stage human heart failure.

    Perrino C, Schroder JN, Lima B, Villamizar N, Nienaber JJ, Milano CA and Naga Prasad SV

    Department of Medicine, Duke University Medical Center, Durham, NC, USA.

    Background: Downregulation of beta-adrenergic receptors (betaARs) under conditions of heart failure requires receptor targeting of phosphoinositide 3-kinase (PI3K)-gamma and redistribution of betaARs into endosomal compartments. Because support with a left ventricular assist device (LVAD) results in significant improvement of cardiac function in humans, we investigated the effects of mechanical unloading on regulation of PI3Kgamma activity and intracellular distribution of betaARs. Additionally, we tested whether displacement of PI3Kgamma from activated betaARs would restore agonist responsiveness in failing human cardiomyocytes.

    To test the role of PI3K on betaAR endocytosis in failing human hearts, we assayed for PI3K activity in human left ventricular samples before and after mechanical unloading (LVAD). Before LVAD, failing human hearts displayed a marked increase in betaAR kinase 1 (betaARK1)-associated PI3K activity that was attributed exclusively to enhanced activity of the PI3Kgamma isoform. Increased betaARK1-coupled PI3K activity in the failing hearts was associated with downregulation of betaARs from the plasma membrane and enhanced sequestration into early and late endosomes compared with unmatched nonfailing controls. Importantly, LVAD support reversed PI3Kgamma activation, normalized the levels of agonist-responsive betaARs at the plasma membrane, and depleted the betaARs from the endosomal compartments without changing the total number of receptors (sum of plasma membrane and early and late endosome receptors). To test whether the competitive displacement of PI3K from the betaAR complex restored receptor responsiveness, we overexpressed the phosphoinositide kinase domain of PI3K (which disrupts betaARK1/PI3K interaction) in primary cultures of failing human cardiomyocytes. Adenoviral-mediated phosphoinositide kinase overexpression significantly increased basal contractility and rapidly reconstituted responsiveness to beta-agonist.

    Conclusions: These results suggest a novel paradigm in which human betaARs undergo a process of intracellular sequestration that is dynamically reversed after LVAD support. Importantly, mechanical unloading leads to complete reversal in PI3Kgamma and betaARK1-associated PI3K activation. Furthermore, displacement of active PI3K from betaARK1 restores betaAR responsiveness in failing myocytes.

    Funded by: NHLBI NIH HHS: HL072183

    Circulation 2007;116;22;2571-9

  • GRK2 interacts with and phosphorylates Nedd4 and Nedd4-2.

    Sanchez-Perez A, Kumar S and Cook DI

    Bosch Institute, Department of Pathology, University of Sydney, Sydney, NSW 2006, Australia.

    Epithelial Na(+) channels (ENaC) mediate the transport of sodium (Na) across epithelia in the kidney, gut, and lungs and are required for blood pressure regulation. They are inhibited by ubiquitin protein ligases, such as Nedd4 and Nedd4-2, which bind to proline-rich motifs (PY motifs) present in the C-termini of ENaC subunits. Loss of inhibition leads to hypertension. ENaC channels are maintained in the active state by G-protein-coupled receptor kinase 2 (GRK2), an enzyme implicated in the development of essential hypertension. Here, we report that GRK2 interacts not only with ENaC, but also with both Nedd4 and Nedd4-2. Additionally, GRK2 is capable of phosphorylating both Nedd4 and Nedd4-2 at multiple sites. Of possible significance is the phosphorylation of the threonine at position 466 in Nedd4, which is located in the area of the ww3 domain that binds ENaC. These results support and extend the role of GRK2 in sodium transport regulation.

    Biochemical and biophysical research communications 2007;359;3;611-5

  • Regulation of beta-adrenergic receptor signaling by S-nitrosylation of G-protein-coupled receptor kinase 2.

    Whalen EJ, Foster MW, Matsumoto A, Ozawa K, Violin JD, Que LG, Nelson CD, Benhar M, Keys JR, Rockman HA, Koch WJ, Daaka Y, Lefkowitz RJ and Stamler JS

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

    beta-adrenergic receptors (beta-ARs), prototypic G-protein-coupled receptors (GPCRs), play a critical role in regulating numerous physiological processes. The GPCR kinases (GRKs) curtail G-protein signaling and target receptors for internalization. Nitric oxide (NO) and/or S-nitrosothiols (SNOs) can prevent the loss of beta-AR signaling in vivo, but the molecular details are unknown. Here we show in mice that SNOs increase beta-AR expression and prevent agonist-stimulated receptor downregulation; and in cells, SNOs decrease GRK2-mediated beta-AR phosphorylation and subsequent recruitment of beta-arrestin to the receptor, resulting in the attenuation of receptor desensitization and internalization. In both cells and tissues, GRK2 is S-nitrosylated by SNOs as well as by NO synthases, and GRK2 S-nitrosylation increases following stimulation of multiple GPCRs with agonists. Cys340 of GRK2 is identified as a principal locus of inhibition by S-nitrosylation. Our studies thus reveal a central molecular mechanism through which GPCR signaling is regulated.

    Funded by: NHLBI NIH HHS: P01-HL075443, R01 HL16037, R01 HL61690, R01 HL70631; NIEHS NIH HHS: U19-ES012496

    Cell 2007;129;3;511-22

  • Lymphocyte G-protein-coupled receptor kinase-2 is upregulated in patients with Alzheimer's disease.

    Leosco D, Fortunato F, Rengo G, Iaccarino G, Sanzari E, Golino L, Zincarelli C, Canonico V, Marchese M, Koch WJ and Rengo F

    Dipartimento di Medicina Clinica, Scienze Cardiovascolari ed Immunologiche, Università Federico II, Via Pansini 5, Edificio 2, 80131 Naples, Italy. dleosco@unina.it

    Alterations in signal transduction pathway of G-protein-coupled receptors (GPCRs) have been found in the cerebrocortex and in the peripheral cultured tissues of patients with Alzheimer's disease (AD). The G-protein-coupled receptor kinase-2 (GRK2) plays an important role in regulating the GPCRs signaling: its increased expression is associated with receptor desensitization. The aim of this study was to explore GRK2 levels in peripheral lymphocytes of AD patients and to establish a correlation between lymphocyte protein concentrations and the degree of cognitive impairment. GRK2 mRNA and protein expression were evaluated in the lymphocytes of AD patients with mild or moderate/severe cognitive impairment and in age-matched healthy subjects. Both GRK2 mRNA and protein expression were higher in AD patients lymphocytes compared to controls. Furthermore, lymphocyte GRK2 levels were significantly correlated to the degree of cognitive decline. Our preliminary data suggest that GRK2 is involved in GPCRs coupling dysfunction observed in AD patients. Further studies are needed in order to verify whether the lymphocyte GRK2 might be utilized as a novel biomarker in AD diagnosis and clinical monitoring.

    Neuroscience letters 2007;415;3;279-82

  • Vascular dysfunction in human and rat cirrhosis: role of receptor-desensitizing and calcium-sensitizing proteins.

    Hennenberg M, Trebicka J, Biecker E, Schepke M, Sauerbruch T and Heller J

    Department of Internal Medicine I, University of Bonn, Bonn, Germany. Martin.Hennenberg@ukb.uni-bonn.de

    Unlabelled: In cirrhosis, vascular hypocontractility leads to vasodilation and contributes to portal hypertension. Impaired activation of contractile pathways contributes to vascular hypocontractility. Angiotensin II type 1 receptors (AT1-Rs) are coupled to the contraction-mediating RhoA/Rho-kinase pathway and may be desensitized by phosphorylation through G-protein-coupled receptor kinases (GRKs) and binding of beta-arrestin-2. In the present study, we analyzed vascular hypocontractility to angiotensin II in cirrhosis. Human hepatic arteries were obtained during liver transplantation. In rats, cirrhosis was induced by bile duct ligation (BDL). Contractility of rat aortic rings was measured myographically. Protein expression and phosphorylation were analyzed by Western blot analysis. Immunoprecipitation was performed with protein A-coupled Sepharose beads. Myosin light chain (MLC) phosphatase activity was assessed as dephosphorylation of MLCs. Aortas from BDL rats were hyporeactive to angiotensin II and extracellular Ca2+. Expression of AT1-R and Galphaq/11,12,13 remained unchanged in hypocontractile rat and human vessels, whereas GRK-2 and beta-arrestin-2 were up-regulated. The binding of beta-arrestin-2 to the AT1-R was increased in hypocontractile rat and human vessels. Inhibition of angiotensin II-induced aortic contraction by the Rho-kinase inhibitor Y-27632 was pronounced in BDL rats. Basal phosphorylation of the ROK-2 substrate moesin was reduced in vessels from rats and patients with cirrhosis. Analysis of the expression and phosphorylation of Ca(2+)-sensitizing proteins (MYPT1 and CPI-17) in vessels from rats and patients with cirrhosis suggested decreased Ca2+ sensitivity. Angiotensin II-stimulated moesin phosphorylation was decreased in aortas from BDL rats. MLC phosphatase activity was elevated in aortas from BDL rats.

    Conclusion: Vascular hypocontractility to angiotensin II in cirrhosis does not result from changes in expression of AT1-Rs or G-proteins. Our data suggest that in cirrhosis-induced vasodilation, the AT1-R is desensitized by GRK-2 and beta-arrestin-2 and that changed patterns of phosphorylated Ca(2+) sensitizing proteins decrease Ca(2+) sensitivity.

    Hepatology (Baltimore, Md.) 2007;45;2;495-506

  • G protein-coupled receptor kinase 2-mediated phosphorylation of downstream regulatory element antagonist modulator regulates membrane trafficking of Kv4.2 potassium channel.

    Ruiz-Gomez A, Mellström B, Tornero D, Morato E, Savignac M, Holguín H, Aurrekoetxea K, González P, González-García C, Ceña V, Mayor F and Naranjo JR

    Departamento de Biología Molecular and Centro de Biología Molecular Severo Ochoa Universidad Autonoma de Madrid-Consejo Superior de Investigaciones Científicas, Campus de Cantoblanco, 28049 Madrid, Spain.

    Downstream regulatory element antagonist modulator (DREAM)/potassium channel interacting protein (KChIP3) is a multifunctional protein of the neuronal calcium sensor subfamily of Ca2+-binding proteins with specific roles in different cell compartments. In the nucleus, DREAM acts as a Ca2+-dependent transcriptional repressor, and outside the nucleus DREAM interacts with Kv4 potassium channels, regulating their trafficking to the cell membrane and their gating properties. In this study we characterized the interaction of DREAM with GRK6 and GRK2, members of the G protein-coupled receptor kinase family of proteins, and their phosphorylation of DREAM. Ser-95 was identified as the site phosphorylated by GRK2. This phosphorylation did not modify the repressor activity of DREAM. Mutation of Ser-95 to aspartic acid, however, blocked DREAM-mediated membrane expression of the Kv4.2 potassium channel without affecting channel tetramerization. Treatment with the calcineurin inhibitors FK506 and cyclosporin A also blocked DREAM-mediated Kv4.2 channel trafficking and calcineurin de-phosphorylated GRK2-phosphorylated DREAM in vitro. Our results indicate that these two Ca2+-dependent posttranslational events regulate the activity of DREAM on Kv4.2 channel function.

    The Journal of biological chemistry 2007;282;2;1205-15

  • Identification of G-protein coupled receptor kinase 2 in paired helical filaments and neurofibrillary tangles.

    Takahashi M, Uchikado H, Caprotti D, Weidenheim KM, Dickson DW, Ksiezak-Reding H and Pasinetti GM

    Neuroinflammation Research Laboratories, Department of Psychiatry, Mount Sinai School of Medicine and the James J. Peters Veteran Affairs Medical Center, 130 West Kingsbridge Road, Bronx, NY 10468, USA.

    G-protein coupled receptor kinases (GRKs) constitute a serine/threonine kinase family playing a major role in agonist-induced phosphorylation and desensitization of G-protein coupled receptors. Recently, GRK2 and GRK5 have been demonstrated to phosphorylate alpha-synuclein (Ser129) and other synuclein isoforms. We studied colocalization of GRK2, GRK5, alpha-synuclein, and tau in neurodegenerative disorders characterized by fibrillary tau inclusions and/or alpha-synuclein-enriched Lewy bodies. We found that Lewy bodies were negative for both GRK2 and GRK5 in Lewy body disease (LBD) and LBD mixed with Alzheimer disease (AD + LBD). Instead, GRK2 but not GRK5 colocalized with 40% to 50% of neurofibrillary tangles in AD + LBD and AD brains. In disorders with less prominent alpha-synucleinopathy, neuronal and glial fibrillary tau deposits known to contain distinct subsets of tau isoforms were also positive for GRK2. These deposits included tufted astrocytes and coiled bodies in progressive supranuclear palsy, astrocytic plaques in corticobasal degeneration, and Pick bodies in Pick disease. In addition, paired helical filaments isolated from AD and AD + LBD brains were found to immunogold-label for GRK2, suggesting that GRK2 could be a potential tau kinase associated with fibrillary tau. Our studies indicate that GRK2 is a novel component of neuronal and glial fibrillary tau deposits with no preference in tau isoform binding. GRK2 may play a role in hyperphosphorylation of tau in tauopathies.

    Journal of neuropathology and experimental neurology 2006;65;12;1157-69

  • G protein-coupled receptor kinase and beta-arrestin-mediated desensitization of the angiotensin II type 1A receptor elucidated by diacylglycerol dynamics.

    Violin JD, Dewire SM, Barnes WG and Lefkowitz RJ

    Department of Medicine, Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, USA.

    Receptor desensitization progressively limits responsiveness of cells to chronically applied stimuli. Desensitization in the continuous presence of agonist has been difficult to study with available assay methods. Here, we used a fluorescence resonance energy transfer-based live cell assay for the second messenger diacylglycerol to measure desensitization of a model seven-transmembrane receptor, the Gq-coupled angiotensin II type 1(A) receptor, expressed in human embryonic kidney 293 cells. In response to angiotensin II, we observed a transient diacylglycerol response reflecting activation and complete desensitization of the receptor within 2-5 min. By utilizing a variety of approaches including graded tetracycline-inducible receptor expression, mutated receptors, and overexpression or short interfering RNA-mediated silencing of putative components of the cellular desensitization machinery, we conclude that the rate and extent of receptor desensitization are critically determined by the following: receptor concentration in the plasma membrane; the presence of phosphorylation sites on the carboxyl terminus of the receptor; kinase activity of G protein-coupled receptor kinase 2, but not of G protein-coupled receptor kinases 3, 5, or 6; and stoichiometric expression of beta-arrestin. The findings introduce the use of the biosensor diacylglycerol reporter as a powerful means for studying Gq-coupled receptor desensitization and document that, at the levels of receptor overexpression commonly used in such studies, the properties of the desensitization process are markedly perturbed and do not reflect normal cellular physiology.

    Funded by: NHLBI NIH HHS: HL16037, HL70631

    The Journal of biological chemistry 2006;281;47;36411-9

  • Impaired neutrophil chemotaxis in sepsis associates with GRK expression and inhibition of actin assembly and tyrosine phosphorylation.

    Arraes SM, Freitas MS, da Silva SV, de Paula Neto HA, Alves-Filho JC, Auxiliadora Martins M, Basile-Filho A, Tavares-Murta BM, Barja-Fidalgo C and Cunha FQ

    Department of Pharmacology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Avenida Bandeirantes, 3900, Ribeirão Preto, São Paulo, Brazil 14049-900.

    The deregulation of inflammatory response during sepsis seems to reflect the overproduction of mediators, which suppress leukocyte functions. We investigated the intracellular mechanisms underlying the inability of neutrophils from severe septic patients to migrate toward chemoattractants. Patients with sepsis (52) and 15 volunteers were prospectively enrolled. Patients presented increased circulating levels of tumor necrosis factor-alpha, interferon-gamma, interleukin (IL)-8, and IL-10. Patients showed reduced neutrophil chemotaxis to formyl-methionyl-leucyl-phenylalanine (FMLP), leukotriene B4 (LTB4) or IL-8. No difference in the transcription or expression of the IL-8 receptor, CXCR1, was detected in neutrophils from controls and patients. However, septic neutrophils failed to increase tyrosine phosphorylation and actin polymerization in response to IL-8 or LTB4. In contrast, septic neutrophils, similar to controls, showed phagocytic activity that induced actin polymerization and augmented phosphotyrosine content. Treatment of control neutrophils with cytokines and lipopolysaccharide (LPS) to mimic endogenous septic environment inhibited actin polymerization and tyrosine phosphorylation in response to IL-8 or LTB4. High expression of G protein-coupled receptor kinase 2 (GRK2) and GRK5 was detected in septic neutrophils and control cells treated with cytokines plus LPS. Data suggest that endogenous mediators produced during sepsis might continually activate circulating neutrophils, leading to GRK activation, which may induce neutrophil desensitization to chemoattractants.

    Blood 2006;108;9;2906-13

  • Tyrosine phosphorylation of G-protein-coupled-receptor kinase 2 (GRK2) by c-Src modulates its interaction with Galphaq.

    Mariggiò S, García-Hoz C, Sarnago S, De Blasi A, Mayor F and Ribas C

    Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Via Nazionale 8A, 66030 Santa Maria Imbaro, Chieti, Italy.

    G-protein-coupled-receptor kinase 2 (GRK2) plays a key role in the modulation of G-protein-coupled-receptor (GPCR) signaling by both phosphorylating agonist-occupied GPCRs and by directly binding to activated Galphaq subunits, inhibiting downstream effectors activation. The GRK2/Galphaq interaction involves the N-terminal region of the kinase that displays homology to regulators of G-protein signaling (RGS) proteins. We have previously reported that upon GPCR stimulation, GRK2 can be phosphorylated by c-Src on tyrosine residues that are present in the RGS-homology (RH) region of this kinase. Here, we demonstrate that c-Src kinase activity increases the interaction between GRK2 and Galphaq. Tyrosine phosphorylation of GRK2 appears to be critically involved in the modulation of this interaction since the stimulatory effect of c-Src is not observed with a GRK2 mutant with impaired tyrosine phosphorylation (GRK2 Y13,86,92F), whereas a mutant that mimics GRK2 tyrosine phosphorylation in these residues displays an increased interaction with Galphaq. As evidence for a physiological role of this modulatory mechanism, activation of the muscarinic receptor M1, a Galphaq-coupled receptor, promotes an increase in GRK2/Galphaq co-immunoprecipitation that parallels the enhanced GRK2 phosphorylation on tyrosine residues. Moreover, c-Src activation enhances inhibition of the Galphaq/phospholipase Cbeta signaling pathway in intact cells, in a GRK2-tyrosine-phosphorylation-dependent manner. Our results suggest a feedback mechanism by which phosphorylation of GRK2 by c-Src increases both GRK2 kinase activity towards GPCRs and its specific interaction with Galphaq subunits, leading to a more rapid switch off of Galphaq-mediated signaling.

    Cellular signalling 2006;18;11;2004-12

  • Phosphorylation of p38 by GRK2 at the docking groove unveils a novel mechanism for inactivating p38MAPK.

    Peregrin S, Jurado-Pueyo M, Campos PM, Sanz-Moreno V, Ruiz-Gomez A, Crespo P, Mayor F and Murga C

    Departamento de Biología Molecular and Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, 28049 Madrid, Spain.

    p38 Mitogen-activated protein kinases (MAPK) are a family of Ser/Thr kinases that regulate important cellular processes such as stress responses, differentiation, and cell-cycle control . Activation of MAPK is achieved through a linear signaling cascade in which upstream kinases (MAPKKs) dually phosphorylate MAPKs at a conserved 3-amino-acid motif (Thr-X-Tyr) . G-protein-coupled receptor kinases (GRKs) are known to selectively phosphorylate G-protein-coupled receptors (GPCRs) and thus trigger desensitization . We report that GRK2 is a novel inactivating kinase of p38MAPK. p38 associates with GRK2 endogenously and is phosphorylated by GRK2 at Thr-123, a residue located at its docking groove. Mimicking phosphorylation at this site impairs the binding and activation of p38 by MKK6 and diminishes the capacity of p38 to bind and phosphorylate its substrates. Accordingly, p38 activation is decreased or increased when cellular GRK2 levels are enhanced or reduced, respectively. Changes in GRK2 levels and activity can modify p38-dependent processes such as differentiation of preadipocytic cells and LPS-induced cytokine release, enhanced in macrophages from GRK2(+/-) mice. Phosphorylation of p38 at a region key for its interaction with different partners uncovers a new mechanism for the regulation of this important family of kinases.

    Current biology : CB 2006;16;20;2042-7

  • Clathrin required for phosphorylation and internalization of beta2-adrenergic receptor by G protein-coupled receptor kinase 2 (GRK2).

    Mangmool S, Haga T, Kobayashi H, Kim KM, Nakata H, Nishida M and Kurose H

    Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Fukuoka 812-8582, Japan.

    Clathrin is a major component of clathrin-coated pits and serves as a binding scaffold for endocytic machinery through the binding of a specific sequence known as the clathrin-binding motif. This motif is also found in cellular signaling proteins other than endocytic components, including G protein-coupled receptor kinase 2 (GRK2), which phosphorylates G protein-coupled receptors and promotes uncoupling of receptor-G protein interaction. However, the functions of clathrin in the regulation of GRK2 are unknown. Here we demonstrated that overexpression of GRK2 mutated at the clathrin-binding motif with alanine (GRK2-5A) results in inhibition of phosphorylation and internalization of the beta2-adrenergic receptor (beta2AR). However, the interaction of beta2AR with GRK2-5A is the same as that of wild type GRK2 as determined by bioluminescence resonance energy transfer. Furthermore, GRK2-5A phosphorylates rhodopsin essentially to the same extent as wild type GRK2 in vitro. Depletion of the clathrin heavy chain using small interference RNA inhibits agonist-induced phosphorylation and internalization of beta2AR. Thus, clathrin works as a regulator of GRK2 in cells. These results indicate that clathrin is a novel player in cellular functions in addition to being a component of endocytosis.

    The Journal of biological chemistry 2006;281;42;31940-9

  • Mdm2 is involved in the ubiquitination and degradation of G-protein-coupled receptor kinase 2.

    Salcedo A, Mayor F and Penela P

    Departamento de Biología Molecular and Centro de Biología Molecular 'Severo Ochoa', Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain.

    G-protein-coupled receptor kinase 2 (GRK2) is a central regulator of G-protein-coupled receptor signaling. We report that Mdm2, an E3-ubiquitin ligase involved in the control of cell growth and apoptosis, plays a key role in GRK2 degradation. Mdm2 and GRK2 association is enhanced by beta(2)-adrenergic receptor stimulation and beta-arrestin. Increased Mdm2 expression accelerates GRK2 proteolysis and promotes kinase ubiquitination at defined residues, whereas GRK2 turnover is markedly impaired in Mdm2-deficient cells. Moreover, we find that activation of the PI3K/Akt pathway by insulin-like growth factor-1 alters Mdm2-mediated GRK2 degradation, leading to enhanced GRK2 stability and increased kinase levels. These data put forward a novel mechanism for controlling GRK2 expression in physiological and pathological conditions.

    The EMBO journal 2006;25;20;4752-62

  • Decreased expression of G protein-coupled receptor kinases in the detrusor smooth muscle of human urinary bladder with outlet obstruction.

    Furuya Y, Araki I, Kamiyama M, Zakoji H, Takihana Y and Takeda M

    Department of Urology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Yamanashi, Japan. yfuruya@res.yamanashi-med.ac.jp

    Aim: We examine the expression of mRNA of G protein-coupled receptor kinase (GRK) subtypes and muscarinic acetylcholine receptor (M) subtypes in the detrusor smooth muscle of the human urinary bladder. Furthermore, we confirm the presence and the localization of GRK proteins in the detrusor smooth muscle of the obstructed bladder in comparison with the control bladder.

    Methods: Detrusor smooth muscle tissues of the human urinary bladder were obtained from 12 male patients; 6 patients did not have bladder outlet obstruction, and the other 6 patients had bladder outlet obstruction. Portions of the dome or anterior wall of the urinary bladder were used for the present study. Reverse transcription/polymerase chain reaction for GRK2, M2 and M3 was performed using total RNA extracted from human urinary bladder detrusor. Antibodies to GRK2, GRK3 and GRK4 were used to confirm the presence of the protein product in the human urinary bladder using immunohistochemical staining and the western blotting technique.

    Results: All complementary DNA (cDNA) transcribed from three different mRNA (M2, M3 and GRK2) were successfully amplified and size-fractionated. The expression of GRK2 protein was strong in the human bladder detrusor, but was significantly weakened by western blotting in obstructed bladder in comparison with control bladder.

    Conclusions: Failure in desensitization mechanisms of muscarinic acetylcholine receptors might be related to storage symptom elicited by overactivity in obstructed bladder with benign prostatic hyperplasia.

    International journal of urology : official journal of the Japanese Urological Association 2006;13;9;1226-32

  • Overexpression of GRK2 in Alzheimer disease and in a chronic hypoperfusion rat model is an early marker of brain mitochondrial lesions.

    Obrenovich ME, Smith MA, Siedlak SL, Chen SG, de la Torre JC, Perry G and Aliev G

    Department of Pathology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106, USA.

    Heterotrimeric guanine nucleotide-binding (G) protein-coupled receptor kinases (GRKs) are cytosolic proteins that are known to contribute to the adaptation of the heptahelical G protein-coupled receptors (GPCRs) and to regulate downstream signals through these receptors. GPCRs mediate the action of messengers that are key modulators of cardiac and vascular cell function, such as growth and differentiation. GRKs are members of a multigene family, which are classified into three subfamilies and are found in cardiac, vascular and cerebral tissues. Increasing evidence strongly supports the hypothesis that vascular damage is an early contributor to the development of Alzheimer disease (AD) and/or other pathology that can mimic human AD. Based on this hypothesis, and since kinases of this family are known to regulate numerous receptor functions both in the brain, myocardium and elsewhere, we explored cellular and subcellular localization by immunoreactivity of G protein-coupled receptor kinase 2 (GRK2), also known as beta-adrenergic receptor kinase-1(betaARK1), in the early pathogenesis of AD and in ischemia reperfusion injury models of brain hypoperfusion. In the present study, we used the two-vessel carotid artery occlusion model, namely the 2-VO system that results in chronic brain hypoperfusion (CBH) and mimics mild cognitive impairment (MCI) and vascular changes in AD pathology. Our findings demonstrate the early overexpression of GRK2 member kinase in the cerebrovasculature, especially endothelial cells (EC) following CBH, as well as in select cells from human AD tissue. We found a significant increase in GRK2 immunoreactivity in the EC of AD patients and after CBH, which preceded any amyloid deposition. Since GRK2 activity is associated with certain compensatory changes in brain cellular compartments and in ischemic cardiac tissue, our findings suggest that chronic hypoperfusion initiates oxidative stress in these conditions and appears to be the main initiating injury stimulus for disruption of brain and cerebrovascular homeostasis and metabolism.

    Neurotoxicity research 2006;10;1;43-56

  • Alpha2A- and alpha2C-adrenergic receptors form homo- and heterodimers: the heterodimeric state impairs agonist-promoted GRK phosphorylation and beta-arrestin recruitment.

    Small KM, Schwarb MR, Glinka C, Theiss CT, Brown KM, Seman CA and Liggett SB

    Department of Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA.

    Dimerization of seven transmembrane-spanning receptors diversifies their pharmacologic and physiologic properties. The alpha(2)-adrenergic receptor (alpha(2)AR) subtypes A and C are both expressed on presynaptic nerves and act to inhibit norepinephrine release via negative feedback. However, in vivo and in vitro studies examining the roles of the two individual alpha(2A)- and alpha(2C)AR subtypes are not readily reconciled. We tested the hypothesis that the receptors form homo- and heterodimers and that the alpha(2A)-alpha(2C) heterodimer has unique properties. SDS-PAGE of epitope-tagged receptors revealed potential oligomers including dimers. BRET of live HEK-293 cells transfected with the subtypes fused to Rluc or YFP revealed that both subtypes form dimers and the heterodimer. A lower BRET(50) for the alpha(2A)-alpha(2C) heterodimer (0.79 +/- 0.20) compared to that of the alpha(2A) or alpha(2C) homodimer (2.331 +/- 0.44 or 3.67 +/- 0.69, respectively) suggests that when both subtypes are expressed, there is a greater likelihood that the two receptors will form the heterodimer than homodimers. Co-immunoprecipitation studies confirmed homo- and heterodimer formation. The presence of the alpha(2C)AR within the heterodimer resulted in a marked reduction in the level of GRK2-mediated alpha(2A)AR phosphorylation, which was accompanied by a qualitative attenuation of beta-arrestin recruitment. Signaling of the alpha(2A)-alpha(2C) heterodimer to the beta-arrestin-dependent activation of Akt was decreased compared to that of the alpha(2A)AR homodimer, while p44/p42 MAP kinase activation was unaffected. Thus, the alpha(2C)AR alters alpha(2A)AR signaling by forming oligomers, and these complexes, which appear to be preferred over the homodimers, should be considered a functional signaling unit in cells in which both subtypes are expressed.

    Funded by: NHLBI NIH HHS: HL045967, HL071609

    Biochemistry 2006;45;15;4760-7

  • Phosphodiesterase-4 influences the PKA phosphorylation status and membrane translocation of G-protein receptor kinase 2 (GRK2) in HEK-293beta2 cells and cardiac myocytes.

    Li X, Huston E, Lynch MJ, Houslay MD and Baillie GS

    Molecular Pharmacology Group, Division of Biochemistry & Molecular Biology, IBLS, Wolfson Link Building, University of Glasgow, University Avenue, Glasgow G12 8QQ, Scotland, UK.

    Membrane-recruitment of GRK2 (G-protein receptor kinase 2) provides a fundamental step in the desensitization process controlling GPCRs (G-protein-coupled receptors), such as the beta2AR (beta2-adrenergic receptor). In the present paper, we show that challenge of HEK-293beta2 [human embryonic kidney cells stably overexpressing the FLAG-tagged beta2AR-GFP (green fluorescent protein)] cells with the beta-adrenoceptor agonist, isoprenaline, causes GRK2 to become phosphorylated by PKA (cAMP-dependent protein kinase). This action is facilitated when cAMP-specific PDE4 (phosphodiesterase-4) activity is selectively inactivated, either chemically with rolipram or by siRNA (small interfering RNA)-mediated knockdown of PDE4B and PDE4D. PDE4-selective inhibition by rolipram facilitates the isoprenaline-induced membrane translocation of GRK2, phosphorylation of the beta2AR by GRK2, membrane translocation of beta-arrestin and internalization of beta2ARs. PDE4-selective inhibition also enhances the ability of isoprenaline to trigger the PKA phosphorylation of GRK2 in cardiac myocytes. In the absence of isoprenaline, rolipram-induced inhibition of PDE4 activity in HEK-293beta2 cells acts to stimulate PKA phosphorylation of GRK2, with consequential effects on GRK2 membrane recruitment and GRK2-mediated phosphorylation of the beta2AR. We propose that a key role for PDE4 enzymes is: (i) to gate the action of PKA on GRK2, influencing the rate of GRK2 phosphorylation of the beta2AR and consequential recruitment of beta-arrestin subsequent to beta-adrenoceptor agonist challenge, and (ii) to protect GRK2 from inappropriate membrane recruitment in unstimulated cells through its phosphorylation by PKA in response to fluctuations in basal levels of cAMP.

    Funded by: Medical Research Council: G8604010; Wellcome Trust

    The Biochemical journal 2006;394;Pt 2;427-35

  • Towards a proteome-scale map of the human protein-protein interaction network.

    Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N, Klitgord N, Simon C, Boxem M, Milstein S, Rosenberg J, Goldberg DS, Zhang LV, Wong SL, Franklin G, Li S, Albala JS, Lim J, Fraughton C, Llamosas E, Cevik S, Bex C, Lamesch P, Sikorski RS, Vandenhaute J, Zoghbi HY, Smolyar A, Bosak S, Sequerra R, Doucette-Stamm L, Cusick ME, Hill DE, Roth FP and Vidal M

    Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA.

    Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

    Funded by: NCI NIH HHS: R33 CA132073; NHGRI NIH HHS: P50 HG004233, R01 HG001715, RC4 HG006066, U01 HG001715; NHLBI NIH HHS: U01 HL098166

    Nature 2005;437;7062;1173-8

  • Chemical genetic engineering of G protein-coupled receptor kinase 2.

    Kenski DM, Zhang C, von Zastrow M and Shokat KM

    Chemistry and Chemical Biology Graduate Program, Department of Psychiatry, University of California, San Francisco, California 94143, USA.

    G protein-coupled receptor kinases (GRKs) play a pivotal role in receptor regulation. Efforts to study the acute effects of GRKs in intact cells have been limited by a lack of specific inhibitors. In the present study we have developed an engineered version of GRK2 that is specifically and reversibly inhibited by the substituted nucleotide analog 1-naphthyl-PP1 (1Na-PP1), and we explored GRK2 function in regulated internalization of the mu-opioid receptor (muOR). A previously described method that conferred analog sensitivity on various kinases, by introducing a space-creating mutation in the conserved active site, failed when applied to GRK2 because the corresponding mutation (L271G) rendered the mutant kinase (GRK2-as1) catalytically inactive. A sequence homology-based approach was used to design second-site suppressor mutations. A C221V second-site mutation produced a mutant kinase (GRK2-as5) with full functional activity and analog sensitivity as compared with wild-type GRK2 in vitro and in intact cells. The role of GRK2-as5 activity in the membrane trafficking of the muOR was also characterized. Morphine-induced internalization was completely blocked when GRK2-as5 activity was inhibited before morphine application. However, inhibition of GRK2-as5 during recycling and reinternalization of the muOR did not attenuate these processes. These results suggest there is a difference in the GRK requirement for initial ligand-induced internalization of a G protein-coupled receptor compared with subsequent rounds of reinternalization.

    Funded by: NIAID NIH HHS: AI44009

    The Journal of biological chemistry 2005;280;41;35051-61

  • The G protein-coupled receptor kinase-2 is a TGFbeta-inducible antagonist of TGFbeta signal transduction.

    Ho J, Cocolakis E, Dumas VM, Posner BI, Laporte SA and Lebrun JJ

    Hormones and Cancer Research Unit, Department of Medicine, Royal Victoria Hospital, McGill University, Montreal, Canada.

    Signaling from the activin/transforming growth factor beta (TGFbeta) family of cytokines is a tightly regulated process. Disregulation of TGFbeta signaling is often the underlying basis for various cancers, tumor metastasis, inflammatory and autoimmune diseases. In this study, we identify the protein G-coupled receptor kinase 2 (GRK2), a kinase involved in the desensitization of G protein-coupled receptors (GPCR), as a downstream target and regulator of the TGFbeta-signaling cascade. TGFbeta-induced expression of GRK2 acts in a negative feedback loop to control TGFbeta biological responses. Upon TGFbeta stimulation, GRK2 associates with the receptor-regulated Smads (R-Smads) through their MH1 and MH2 domains and phosphorylates their linker region. GRK2 phosphorylation of the R-Smads inhibits their carboxyl-terminal, activating phosphorylation by the type I receptor kinase, thus preventing nuclear translocation of the Smad complex, leading to the inhibition of TGFbeta-mediated target gene expression, cell growth inhibition and apoptosis. Furthermore, we demonstrate that GRK2 antagonizes TGFbeta-induced target gene expression and apoptosis ex vivo in primary hepatocytes, establishing a new role for GRK2 in modulating single-transmembrane serine/threonine kinase receptor-mediated signal transduction.

    The EMBO journal 2005;24;18;3247-58

  • Protein kinase activity of phosphoinositide 3-kinase regulates beta-adrenergic receptor endocytosis.

    Naga Prasad SV, Jayatilleke A, Madamanchi A and Rockman HA

    Division of Cardiology, Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA.

    Phosphoinositide 3-kinase (PI(3)K) is a unique enzyme characterized by both lipid and protein kinase activities. Here, we demonstrate a requirement for the protein kinase activity of PI(3)K in agonist-dependent beta-adrenergic receptor (betaAR) internalization. Using PI(3)K mutants with either protein or lipid phosphorylation activity, we identify the cytoskeletal protein non-muscle tropomyosin as a substrate of PI(3)K, which is phosphorylated in a wortmannin-sensitive manner on residue Ser 61. A constitutively dephosphorylated (S61A) tropomyosin mutant blocks agonist-dependent betaAR internalization, whereas a tropomyosin mutant that mimics constitutive phosphorylation (S61D) complements the PI(3)K mutant, with only lipid phosphorylation activity reversing the defective betaAR internalization. Notably, knocking down endogenous tropomyosin expression using siRNAs that target different regions if tropomyosin resulted in complete inhibition of betaAR endocytosis, showing that non-muscle tropomyosin is essential for agonist-mediated receptor internalization. These studies demonstrate a previously unknown role for the protein phosphorylation activity of PI(3)K in betaAR internalization and identify non-muscle tropomyosin as a cellular substrate for protein kinase activity of PI(3)K.

    Funded by: NHLBI NIH HHS: P01 HL 075443

    Nature cell biology 2005;7;8;785-96

  • Regulation of EGF-induced ERK/MAPK activation and EGFR internalization by G protein-coupled receptor kinase 2.

    Gao J, Li J and Ma L

    Pharmacology Research Center, Shanghai Medical College, Fudan University, Shanghai 200032, China. gaojingxia@mail.tongji.edu.cn

    G protein-coupled receptor kinases (GRKs) mediate agonist-induced phosphorylation and desensitization of various G protein-coupled receptors (GPCRs). We investigate the role of GRK2 on epidermal growth factor (EGF) receptor signaling, including EGF-induced extracellular signal-regulated kinase and mitogen-activated protein kinase (ERK/MAPK) activation and EGFR internalization. Immunoprecipitation and immunofluorescence experiments show that EGF stimulates GRK2 binding to EGFR complex and GRK2 translocating from cytoplasm to the plasma membrane in human embryonic kidney 293 cells. Western blotting assay shows that EGF-induced ERK/MAPK phosphorylation increases 1.9-fold, 1.1-fold and 1.5-fold (P < 0.05) at time point 30, 60 and 120 min, respectively when the cells were transfected with GRK2, suggesting the regulatory role of GRK2 on EGF-induced ERK/MAPK activation. Flow cytometry experiments show that GRK2 overexpression has no effect on EGF-induced EGFR internalization, however, it increases agonist-induced G protein-coupled delta opioid receptor internalization by approximately 40% (P < 0.01). Overall, these data suggest that GRK2 has a regulatory role in EGF-induced ERK/MAPK activation, and that the mechanisms underlying the modulatory role of GRK2 in EGFR and GPCR signaling pathways are somewhat different at least in receptor internalization.

    Acta biochimica et biophysica Sinica 2005;37;8;525-31

  • Uncoupling and endocytosis of 5-hydroxytryptamine 4 receptors. Distinct molecular events with different GRK2 requirements.

    Barthet G, Gaven F, Framery B, Shinjo K, Nakamura T, Claeysen S, Bockaert J and Dumuis A

    CNRS UMR5203, Montpellier, F-34094, France.

    The 5-hydroxytryptamine type 4 receptors (5-HT4Rs) are involved in memory, cognition, feeding, respiratory control, and gastrointestinal motility through activation of a G(s)/cAMP pathway. We have shown that 5-HT4R undergoes rapid and profound homologous uncoupling in neurons. However, no significant uncoupling was observed in COS-7 or HEK293 cells, which expressed either no or a weak concentration of GRK2, respectively. High expression of GRK2 in neurons is likely to be the reason for this difference because overexpression of GRK2 in COS-7 and HEK293 cells reproduced rapid and profound uncoupling of 5-HT4R. We have also shown, for the first time, that GRK2 requirements for uncoupling and endocytosis were very different. Indeed, beta-arrestin/dynamin-dependent endocytosis was observed in HEK293 cells without any need of GRK2 overexpression. In addition to this difference, uncoupling and beta-arrestin/dynamin-dependent endocytosis were mediated through distinct mechanisms. Neither uncoupling nor beta-arrestin/dynamin-dependent endocytosis required the serine and threonine residues localized within the specific C-terminal domains of the 5-HT4R splice variants. In contrast, a cluster of serines and threonines, common to all variants, was an absolute requirement for beta-arrestin/dynamin-dependent receptor endocytosis, but not for receptor uncoupling. Furthermore, beta-arrestin/dynamin-dependent endocytosis and uncoupling were dependent on and independent of GRK2 kinase activity, respectively. These results clearly demonstrate that the uncoupling and endocytosis of 5-HT4R require different GRK2 concentrations and involve distinct molecular events.

    The Journal of biological chemistry 2005;280;30;27924-34

  • Phosphorylation-independent regulation of metabotropic glutamate receptor 1 signaling requires g protein-coupled receptor kinase 2 binding to the second intracellular loop.

    Dhami GK, Babwah AV, Sterne-Marr R and Ferguson SS

    Cell Biology Research Group, Robarts Research Laboratory and Department of Physiology and Pharmacology, The University of Western Ontario, 100 Perth Drive, London, Ontario N6A 5K8, Canada.

    Metabotropic glutamate receptors (mGluRs) are members of a unique class of G protein-coupled receptors (class III) that include the calcium-sensing and gamma-aminobutyric acid type B receptors. The activity of mGluRs is regulated by second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs). The attenuation of both mGluR1a and mGluR1b signaling by GRK2 is phosphorylation- and beta-arrestin-independent and requires the concomitant association of GRK2 with both the receptor and Galpha(q/11). G protein interactions are mediated, in part, by the mGluR1 intracellular second loop, but the domains required for GRK2 binding are unknown. In the present study, we showed that GRK2 binds to the second intracellular loop of mGluR1a and mGluR1b and also to the mGluR1a carboxyl-terminal tail. Alanine scanning mutagenesis revealed a discrete domain within loop 2 that contributes to GRK2 binding, and the mutation of either lysine 691 or 692 to an alanine within this domain resulted in a loss of GRK2 binding to both mGluR1a and mGluR1b. Mutation of either Lys(691) or Lys(692) prevented GRK2-mediated attenuation of mGluR1b signaling, whereas the mutation of only Lys(692) prevented GRK2-mediated inhibition of mGluR1a signaling. Thus, the mGluR1a carboxyl-terminal tail may also be involved in regulating the signaling of the mGluR1a splice variant. Taken together, our findings indicated that kinase binding to an mGluR1 domain involved in G protein-coupling is essential for the phosphorylation-independent attenuation of signaling by GRK2.

    The Journal of biological chemistry 2005;280;26;24420-7

  • G protein-coupled receptor kinase 2-mediated phosphorylation of ezrin is required for G protein-coupled receptor-dependent reorganization of the actin cytoskeleton.

    Cant SH and Pitcher JA

    MRC Laboratory for Molecular and Cellular Biology and Department of Pharmacology, University College London, London, WC1E 6BT United Kingdom.

    G protein-coupled receptor kinase 2 (GRK2) phosphorylates and desensitizes activated G protein-coupled receptors (GPCRs). Here, we identify ezrin as a novel non-GPCR substrate of GRK2. GRK2 phosphorylates glutathione S-transferase (GST)-ezrin, but not an ezrin fusion protein lacking threonine 567 (T567), in vitro. These results suggest that T567, the regulatory phosphorylation site responsible for maintaining ezrin in its active conformation, represents the principle site of GRK2-mediated phosphorylation. Two lines of evidence indicate that GRK2-mediated ezrin-radixinmoesin (ERM) phosphorylation serves to link GPCR activation to cytoskeletal reorganization. First, in Hep2 cells muscarinic M1 receptor (M1MR) activation causes membrane ruffling. This ruffling response is ERM dependent and is accompanied by ERM phosphorylation. Inhibition of GRK2, but not rho kinase or protein kinase C, prevents ERM phosphorylation and membrane ruffling. Second, agonist-induced internalization of the beta2-adrenergic receptor (beta2AR) and M1MR is accompanied by ERM phosphorylation and localization of phosphorylated ERM to receptor-containing endocytic vesicles. The colocalization of internalized beta2AR and phosphorylated ERM is not dependent on Na+/H+ exchanger regulatory factor binding to the beta2AR. Inhibition of ezrin function impedes beta2AR internalization, further linking GPCR activation, GRK activity, and ezrin function. Overall, our results suggest that GRK2 serves not only to attenuate but also to transduce GPCR-mediated signals.

    Funded by: Wellcome Trust

    Molecular biology of the cell 2005;16;7;3088-99

  • G protein-coupled receptor kinase 2 in multiple sclerosis and experimental autoimmune encephalomyelitis.

    Vroon A, Kavelaars A, Limmroth V, Lombardi MS, Goebel MU, Van Dam AM, Caron MG, Schedlowski M and Heijnen CJ

    Laboratory for Psychoneuroimmunology, University Medical Center Utrecht, Utrecht, The Netherlands.

    Many modulators of inflammation, including chemokines, neuropeptides, and neurotransmitters signal via G protein-coupled receptors (GPCR). GPCR kinases (GRK) can phosphorylate agonist-activated GPCR thereby promoting receptor desensitization. Here we describe that in leukocytes from patients with active relapsing-remitting multiple sclerosis (MS) or with secondary progressive MS, GRK2 levels are significantly reduced. Unexpectedly, cells from patients during remission express even lower levels of GRK2. The level of GRK2 in leukocytes of patients after stroke, a neurological disorder with paralysis but without an autoimmune component, was similar to GRK2 levels in cells from healthy individuals. In addition, we demonstrate that the course of recombinant myelin oligodendrocyte glycoprotein (1-125)-induced experimental autoimmune encephalomyelitis (EAE), an animal model for MS, is markedly different in GRK2(+/-) mice that express 50% of the GRK2 protein in comparison with wild-type mice. Onset of EAE was significantly advanced by 5 days in GRK2(+/-) mice. The earlier onset of EAE was associated with increased early infiltration of the CNS by T cells and macrophages. Although disease scores in the first phase of EAE were similar in both groups, GRK2(+/-) animals did not develop relapses, whereas wild-type animals did. The absence of relapses in GRK2(+/-) mice was associated with a marked reduction in inflammatory infiltrates in the CNS. Recombinant myelin oligodendrocyte glycoprotein-induced T cell proliferation and cytokine production were normal in GRK2(+/-) animals. We conclude that down-regulation of GRK2 expression may have important consequences for the onset and progression of MS.

    Journal of immunology (Baltimore, Md. : 1950) 2005;174;7;4400-6

  • Structure/function analysis of alpha2A-adrenergic receptor interaction with G protein-coupled receptor kinase 2.

    Pao CS and Benovic JL

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

    G protein-coupled receptors (GPCRs) mediate the ability of a diverse array of extracellular stimuli to control intracellular signaling. Many GPCRs are phosphorylated by G protein-coupled receptor kinases (GRKs), a process that mediates agonist-specific desensitization in many cells. Although GRK binding to activated GPCRs results in kinase activation and receptor phosphorylation, relatively little is known about the mechanism of GRK/GPCR interaction or how this interaction results in kinase activation. Here, we used the alpha2A-adrenergic receptor (alpha(2A)AR) as a model to study GRK/receptor interaction because GRK2 phosphorylation of four adjacent serines within the large third intracellular loop of this receptor is known to mediate desensitization. Various domains of the alpha(2A)AR were expressed as glutathione S-transferase fusion proteins and tested for the ability to bind purified GRK2. The second and third intracellular loops of the alpha(2A)AR directly interacted with GRK2, whereas the first intracellular loop and C-terminal domain did not. Truncation mutagenesis identified three discrete regions within the third loop that contributed to GRK2 binding, the membrane proximal N- and C-terminal regions as well as a central region adjacent to the phosphorylation sites. Site-directed mutagenesis revealed a critical role for specific basic residues within these regions in mediating GRK2 interaction with the alpha(2A)AR. Mutation of these residues within the holo-alpha(2A)AR diminished GRK2-promoted phosphorylation of the receptor as well as the ability of the kinase to be activated by receptor binding. These studies provide new insight into the mechanism of interaction and activation of GRK2 by GPCRs and suggest that GRK2 binding is critical not only for receptor phosphorylation but also for full activity of the kinase.

    Funded by: NIGMS NIH HHS: GM44944

    The Journal of biological chemistry 2005;280;12;11052-8

  • Bimodal regulation of the human H1 histamine receptor by G protein-coupled receptor kinase 2.

    Iwata K, Luo J, Penn RB and Benovic JL

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

    The H1 histamine receptor (H1HR) is a member of the G protein-coupled receptor superfamily and regulates numerous cellular functions through its activation of the G(q/11) subfamily of heterotrimeric G proteins. Although the H1HR has been shown to undergo desensitization in multiple cell types, the mechanisms underlying the regulation of H1HR signaling are poorly defined. To address this issue, we examined the effects of wild type and mutant G protein-coupled receptor kinases (GRKs) on the phosphorylation and signaling of human H1HR in HEK293 cells. Overexpression of GRK2 promoted H1HR phosphorylation in intact HEK293 cells and completely inhibited inositol phosphate production stimulated by H1HR, whereas GRK5 and GRK6 had lesser effects on H1HR phosphorylation and signaling. Interestingly, catalytically inactive GRK2 (GRK2-K220R) also significantly attenuated H1HR-mediated inositol phosphate production, as did an N-terminal fragment of GRK2 previously characterized as a regulator of G protein signaling (RGS) protein for Galpha(q/11). Disruption of this RGS function in holo-GRK2 by mutation (GRK2-D110A) partially reversed the quenching effect of GRK2, whereas deletion of both the kinase activity and RGS function (GRK2-D110A/K220R) effectively relieved the inhibition of inositol phosphate generation. To evaluate the role of endogenous GRKs on H1HR regulation, we used small interfering RNAs to selectively target GRK2 and GRK5, two of the primary GRKs expressed in HEK293 cells. A GRK2-specific small interfering RNA effectively reduced GRK2 expression and resulted in a significant increase in histamine-promoted calcium flux. In contrast, knockdown of GRK5 expression was without effect on H1HR signaling. These findings demonstrate that GRK2 is the principal kinase mediating H1 histamine receptor desensitization in HEK293 cells and suggest that rapid termination of H1HR signaling is mediated by both the kinase activity and RGS function of GRK2.

    Funded by: NHLBI NIH HHS: HL67663; NIGMS NIH HHS: GM44944

    The Journal of biological chemistry 2005;280;3;2197-204

  • Activation of tyrosine kinase of EGFR induces Gbetagamma-dependent GRK-EGFR complex formation.

    Gao J, Li J, Chen Y and Ma L

    Pharmacology Research Center, Shanghai Medical College, Fudan University, 138 Yi Xue Yuan Road, Shanghai 200032, China.

    This study demonstrated that activation of tyrosine kinase of epidermal growth factor receptor (EGFR) induces its association with G protein-coupled receptor kinase 2 (GRK2). Immunoprecipitation experiments showed that EGF stimulation increased GRK2 binding to EGFR complex in HEK293 cells coexpressing EGFR and GRK2. The EGF-induced GRK2-EGFR complex formation was greatly reduced by perturbation of EGFR and Src tyrosine kinase activity. Furthermore, studies with GRK2 mutants showed that neither catalytic activity nor the N-terminal domain of GRK2 was required for EGF-induced GRK2-EGFR complex formation. However, overexpression of Gbetagamma scavengers blocked EGF-induced formation of GRK2-EGFR complex.

    FEBS letters 2005;579;1;122-6

  • Activity-dependent internalization of smoothened mediated by beta-arrestin 2 and GRK2.

    Chen W, Ren XR, Nelson CD, Barak LS, Chen JK, Beachy PA, de Sauvage F and Lefkowitz RJ

    Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA. w.chen@duke.edu

    Binding of Sonic Hedgehog (Shh) to Patched (Ptc) relieves the latter's tonic inhibition of Smoothened (Smo), a receptor that spans the cell membrane seven times. This initiates signaling which, by unknown mechanisms, regulates vertebrate developmental processes. We find that two molecules interact with mammalian Smo in an activation-dependent manner: G protein-coupled receptor kinase 2 (GRK2) leads to phosphorylation of Smo, and beta-arrestin 2 fused to green fluorescent protein interacts with Smo. These two processes promote endocytosis of Smo in clathrin-coated pits. Ptc inhibits association of beta-arrestin 2 with Smo, and this inhibition is relieved in cells treated with Shh. A Smo agonist stimulated and a Smo antagonist (cyclopamine) inhibited both phosphorylation of Smo by GRK2 and interaction of beta-arrestin 2 with Smo. beta-Arrestin 2 and GRK2 are thus potential mediators of signaling by activated Smo.

    Science (New York, N.Y.) 2004;306;5705;2257-60

  • 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 GM062884-01, R01 GM062884-02, R01 GM062884-03, R01 GM062884-04

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

  • D1 dopamine receptor signaling involves caveolin-2 in HEK-293 cells.

    Yu P, Yang Z, Jones JE, Wang Z, Owens SA, Mueller SC, Felder RA and Jose PA

    Department of Pediatrics, Georgetown University School of Medicine, Washington, D.C., USA. yup@georgetown.edu

    Background: Dopamine receptors in the kidney, especially those belonging to the D1-like receptor family, are important in the regulation of renal function and blood pressure. Because of increasing evidence that G protein-coupled receptors (GPCRs) are associated with caveolae and lipid rafts, we tested the hypothesis that the D1 dopamine receptor (D1R) and signaling molecules are regulated by caveolin in caveolae or lipid rafts.

    Methods: Six experimental approaches were used: (1) construction of tagged human D1Rs (hD1Rs) and transfectants; (2) cell culture [human embryonic kidney (HEK)-293 and immortalized rat renal proximal tubule cells] and biotinylation; (3) cell fractionation by sucrose gradient centrifugation; (4) immunoprecipitation and immunoblotting; (5) immunofluorescence and confocal microscopy; and (6) adenylyl cyclase assays.

    Results: hD1Rs, heterologously expressed in HEK-293 cells, formed protein species with molecular mass ranging from 50 to 250 kD, and were localized in lipid rafts and nonraft plasma membranes. The hD1Rs cofractionated with caveolin-2, G protein subunits, and several signaling molecules. Both exogenously expressed hD1Rs and endogenously expressed rat D1Rs colocalized and coimmunoprecipitated with caveolin-2. A D1R agonist (fenoldopam) increased the amount of caveolin-2beta associated with hD1Rs and activated adenylyl cyclase to a greater extent in lipid rafts than in nonraft plasma membranes. Reduction in the expression of caveolin-2 with antisense oligonucleotides attenuated the stimulatory effect of fenoldopam on cyclic adenosine monophosphate (cAMP) accumulation.

    Conclusion: The majority of hD1Rs are distributed in lipid rafts. Heterologously and endogenously expressed D1Rs in renal cells are associated with and regulated by caveolin-2.

    Funded by: NCI NIH HHS: 2P30-CA-51008; NCRR NIH HHS: 1S10 RR15768-01; NHLBI NIH HHS: HL23081, HL68686, HL74940; NIDDK NIH HHS: DK39308, DK52612

    Kidney international 2004;66;6;2167-80

  • Phosphorylation of the platelet-derived growth factor receptor-beta by G protein-coupled receptor kinase-2 reduces receptor signaling and interaction with the Na(+)/H(+) exchanger regulatory factor.

    Hildreth KL, Wu JH, Barak LS, Exum ST, Kim LK, Peppel K and Freedman NJ

    Department of Medicine (Cardiology), Duke University Medical Center, Durham, North Carolina 27710, USA.

    G protein-coupled receptor kinase-2 (GRK2) can phosphorylate and desensitize the platelet-derived growth factor receptor-beta (PDGFRbeta) in heterologous cellular systems. To determine whether GRK2 regulates the PDGFRbeta in physiologic systems, we examined PDGFRbeta signaling in mouse embryonic fibroblasts from GRK2-null and cognate wild type mice. To discern a mechanism by which GRK2-mediated phosphorylation can desensitize the PDGFRbeta, but not the epidermal growth factor receptor (EGFR), we investigated effects of GRK2-mediated phosphorylation on the association of the PDGFRbeta with the Na(+)/H(+) exchanger regulatory factor (NHERF), a protein shown to potentiate dimerization of the PDGFRbeta, but not the EGFR. Physiologic expression of GRK2 diminished (a) phosphoinositide hydrolysis elicited through the PDGFRbeta but not heterotrimeric G proteins; (b) Akt activation evoked by the PDGFRbeta but not the EGFR; and (c) PDGF-induced tyrosyl phosphorylation of the PDGFRbeta itself. PDGFRbeta desensitization by physiologically expressed GRK2 correlated with a 2.5-fold increase in PDGF-promoted PDGFRbeta seryl phosphorylation. In 293 cells, GRK2 overexpression reduced PDGFRbeta/NHERF association by 60%. This effect was reproduced by S1104D mutation of the PDGFRbeta, which also diminished PDGFRbeta activation and signaling (like the S1104A mutation) to an extent equivalent to that achieved by GRK2-mediated PDGFRbeta phosphorylation. GRK2 overexpression desensitized only the wild type but not the S1104A PDGFRbeta. We conclude that GRK2-mediated PDGFRbeta seryl phosphorylation plays an important role in desensitizing the PDGFRbeta in physiologic systems. Furthermore, this desensitization appears to involve GRK2-mediated phosphorylation of PDGFRbeta Ser(1104), with consequent dissociation of the PDGFRbeta from NHERF.

    Funded by: NHLBI NIH HHS: HL63288, HL64744

    The Journal of biological chemistry 2004;279;40;41775-82

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • Hypertensive left ventricular hypertrophy: relation to beta-adrenergic receptor kinase-1 (betaARK1) in peripheral lymphocytes.

    Park SJ, Choi DJ and Kim CW

    Department of Internal Medicine, College of Medicine, Gyeongsang National University, Jinju, Korea.

    Background: Left ventricular hypertrophy (LVH) is associated with increased cardiovascular risk and altered sympathetic regulation in hypertension.

    Objectives: To determine whether the level of beta-adrenergic receptor kinase-1 (betaARK1) in lymphocytes is related to LVH in patients with hypertension.

    Methods: Forty-nine patients with untreated essential hypertension were recruited to the study and classified into two groups: left ventricular hypertrophy (LVH: left ventricular mass index > or =134 g/m in men and > or =110 g/m in women; ages 52.4 +/- 12.8 years, n = 25) and non-LVH (NLVH: left ventricular mass index < 134 g/m in men and < 110 g/m in women; ages 50.8 +/- 13.1 years, n = 24). Lymphocytes were isolated from patients and quantitative-competitive reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblotting were used to estimate the expression of betaARK1 in the lymphocytes. G-protein-coupled receptor kinase activity was assessed by rhodopsin phosphorylation assay.

    Results: The expression of betaARK1 in lymphocytes was greater in the LVH group than in the NLVH group (0.0069 +/- 0.002 ng compared with 0.0048 +/- 0.0017 ng, P < 0.01) and correlated well with left ventricular mass index (r = 0.527, P < 0.001) and relative wall thickness (r = 0.627, P < 0.001). The concentration of betaARK1 protein in lymphocytes from individuals with LVH was increased two-fold compared with that in the NLVH group (both n = 7). Lymphocyte G-protein-coupled receptor kinase activity from LVH was enhanced 1.7-fold compared with NLVH (1.03 +/- 2.16 and 1.79 +/- 1.87 pmol phosphate/min per mg protein, respectively; P < 0.05, n = 7 for each group).

    Conclusions: The concentration of betaARK1 in lymphocytes is greater in hypertensive individuals with LVH than in those without LVH and parallels the degree of hypertrophy. Generalized alterations in beta-adrenergic signalling, including betaARK1, could be a major contributory factor in the development of LVH in hypertension, and the concentration of betaARK1 in lymphocytes can reflect the development of LVH in a patient with hypertension.

    Journal of hypertension 2004;22;5;1025-32

  • Targeted beta-adrenergic receptor kinase (betaARK1) inhibition by gene transfer in failing human hearts.

    Williams ML, Hata JA, Schroder J, Rampersaud E, Petrofski J, Jakoi A, Milano CA and Koch WJ

    Department of Surgery, Duke University Medical Center, Durham, NC, USA.

    Background: Failing human myocardium is characterized by an attenuated contractile response to beta-adrenergic receptor (betaAR) stimulation due to changes in this signaling cascade, including increased expression and activity of the beta-adrenergic receptor kinase (betaARK1). This leads to desensitization and downregulation of betaARs. Previously, expression of a peptide inhibitor of betaARK1 (betaARKct) has proven beneficial in several animal models of heart failure (HF).

    To test the hypothesis that inhibition of betaARK1 could improve beta-adrenergic signaling and contractile function in failing human myocytes, the betaARKct was expressed via adenovirus-mediated (AdbetaARKct) gene transfer in ventricular myocytes isolated from hearts explanted from 10 patients with end-stage HF undergoing cardiac transplantation. AdbetaARKct also contained the marker gene, green fluorescent protein, and successful gene transfer was confirmed via fluorescence and immunoblotting. Compared with uninfected failing myocytes (control), the velocities of both contraction and relaxation in the AdbetaARKct-treated cells were increased in response to the beta-agonist isoproterenol (contraction: 57.5+/-6.6% versus 37.0+/-4.2% shortening per second, P<0.05; relaxation: 43.8+/-5.5% versus 27.5+/-3.9% lengthening per second, P<0.05). Fractional shortening was similarly enhanced (12.2+/-1.2% versus 8.0+/-0.9%, P<0.05). Finally, adenylyl cyclase activity in response to isoproterenol was also increased in AdbetaARKct-treated myocytes.

    Conclusions: These results demonstrate that as in animal models of HF, expression of the betaARKct can improve contractile function and beta-adrenergic responsiveness in failing human myocytes. Thus, betaARK1 inhibition may represent a therapeutic strategy for human HF.

    Funded by: NHLBI NIH HHS: HL59533, R01 HL072183, R01 HL56205

    Circulation 2004;109;13;1590-3

  • Identification and characterization of phosphorylated proteins in the human pituitary.

    Giorgianni F, Beranova-Giorgianni S and Desiderio DM

    The Charles B. Stout Neuroscience Mass Spectrometry Laboratory, University of Tennessee Health Science Center, Memphis, 38163, USA.

    Post-translational modifications of proteins from the human pituitary gland play an important role in the regulation of different body functions. We report on the application of a liquid chromatography-tandem mass spectrometry (MS/MS) based approach to detect and characterize phosphorylated proteins in a whole human pituitary digest. By combining an immobilized metal affinity column-based enrichment method with MS/MS conditions that favor the neutral loss of phosphoric acid from a phosphorylated precursor ion, we identified several previously undescribed phosphorylated peptides. The identified peptides were matched to the sequences of six pituitary proteins: the human growth hormone, chromogranin A, secretogranin I, 60S ribosomal protein P1 and/or P2, DnaJ homolog subfamily C member 5, and galanin. The phosphorylation sites of these important regulatory proteins were determined by MS/MS and MS(3) analysis.

    Funded by: NCRR NIH HHS: RR 10522, RR 14593; NINDS NIH HHS: NS 42843

    Proteomics 2004;4;3;587-98

  • Identification of phosphoproteins and their phosphorylation sites in the WEHI-231 B lymphoma cell line.

    Shu H, Chen S, Bi Q, Mumby M and Brekken DL

    Protein Chemistry Laboratory, Alliance for Cellular Signaling, University of Texas Southwestern Medical Center, Dallas, TX 75390-9196, USA.

    A major goal of the Alliance for Cellular Signaling is to elaborate the components of signal transduction networks in model cell systems, including murine B lymphocytes. Due to the importance of protein phosphorylation in many aspects of cell signaling, the initial efforts have focused on the identification of phosphorylated proteins. In order to identify serine- and threonine-phosphorylated proteins on a proteome-wide basis, WEHI-231 cells were treated with calyculin A, a serine/threonine phosphatase inhibitor, to induce high levels of protein phosphorylation. Proteins were extracted from whole-cell lysates and digested with trypsin. Phosphorylated peptides were then enriched using immobilized metal affinity chromatography and identified by liquid chromatography-tandem mass spectrometry. A total of 107 proteins and 193 phosphorylation sites were identified using these methods. Forty-two of these proteins have been reported to be phosphorylated, but only some of them have been detected in B cells. Fifty-four of the identified proteins were not previously known to be phosphorylated. The remaining 11 phosphoproteins have previously only been characterized as novel cDNA or genomic sequences. Many of the identified proteins were phosphorylated at multiple sites. The proteins identified in this study significantly expand the repertoire of proteins known to be phosphorylated in B cells. The number of newly identified phosphoproteins indicates that B cell signaling pathways utilizing protein phosphorylation are likely to be more complex than previously appreciated.

    Funded by: NIGMS NIH HHS: U54 GM062114

    Molecular & cellular proteomics : MCP 2004;3;3;279-86

  • G protein-coupled receptor kinase interaction with Hsp90 mediates kinase maturation.

    Luo J and Benovic JL

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

    G protein-coupled receptor kinase 2 (GRK2) is a serine/threonine-specific protein kinase that mediates agonist-dependent phosphorylation of numerous G protein-coupled receptors. In an effort to identify proteins that regulate GRK2 function, we searched for interacting proteins by immunoprecipitation of endogenous GRK2 from HL60 cells. Subsequent analysis by gel electrophoresis and mass spectrometry revealed that GRK2 associates with heat shock protein 90 (Hsp90). GRK2 interaction with Hsp90 was confirmed by co-immunoprecipitation and was effectively disrupted by geldanamycin, an Hsp90-specific inhibitor. Interestingly, geldanamycin treatment of HL60 cells decreased the expression of endogenous GRK2 in a dose- and time-dependent manner, and metabolic labeling demonstrated that geldanamycin rapidly accelerated the degradation of newly synthesized GRK2. The use of various protease inhibitors suggested that GRK2 degradation induced by geldanamycin was predominantly through the proteasome pathway. To test whether Hsp90 plays a general role in regulating GRK maturation, additional GRKs were studied by transient expression in COS-1 cells and subsequent treatment with geldanamycin. These studies demonstrate that GRK3, GRK5, and GRK6 are also stabilized by interaction with Hsp90. Taken together, our work revealed that GRK interaction with heat shock proteins plays an important role in regulating GRK maturation.

    Funded by: NIGMS NIH HHS: GM44944

    The Journal of biological chemistry 2003;278;51;50908-14

  • Protein kinase C switches the Raf kinase inhibitor from Raf-1 to GRK-2.

    Lorenz K, Lohse MJ and Quitterer U

    Institut für Pharmakologie und Toxikologie, Versbacher Strasse 9, D-97078 Würzburg, Germany.

    Feedback inhibition is a fundamental principle in signal transduction allowing rapid adaptation to different stimuli. In mammalian cells, the major feedback inhibitor for G-protein-coupled receptors (GPCR) is G-protein-coupled receptor kinase 2 (GRK-2), which phosphorylates activated receptors, uncouples them from G proteins and initiates their internalization. The functions of GRK-2 are indispensable and need to be tightly controlled. Dysregulation promotes disorders such as hypertension or heart failure. In our search for a control mechanism for this vital kinase, here we show that the Raf kinase inhibitor protein (RKIP) is a physiological inhibitor of GRK-2. After stimulation of GPCR, RKIP dissociates from its known target, Raf-1 (refs 6-8), to associate with GRK-2 and block its activity. This switch is triggered by protein kinase C (PKC)-dependent phosphorylation of the RKIP on serine 153. The data delineate a new principle in signal transduction: by activating PKC, the incoming receptor signal is enhanced both by removing an inhibitor from Raf-1 and by blocking receptor internalization. A physiological role for this mechanism is shown in cardiomyocytes in which the downregulation of RKIP restrains beta-adrenergic signalling and contractile activity.

    Nature 2003;426;6966;574-9

  • Agonist-induced formation of opioid receptor-G protein-coupled receptor kinase (GRK)-G beta gamma complex on membrane is required for GRK2 function in vivo.

    Li J, Xiang B, Su W, Zhang X, Huang Y and Ma L

    National Laboratory of Medical Neurobiology, Shanghai Medical College and Institutes of Brain Science, Fudan University, Shanghai 200032, China.

    G protein-coupled receptor kinases (GRKs) catalyze agonist-induced receptor phosphorylation on the membrane and initiate receptor desensitization. Previous in vitro studies have shown that the binding of GRK to membrane-associated G beta gamma subunits plays an important role in translocation of GRK2 from the cytoplasm to the plasma membrane. The current study investigated the role of the interaction of GRK2 with the activated delta-opioid receptor (DOR) and G beta gamma subunits in the membrane translocation and function of GRK2 using intact human embryonic kidney 293 cells. Our results showed that agonist treatment induced GRK2 binding to DOR, GRK2 translocation to the plasma membrane, and DOR phosphorylation in cells expressing the wild-type DOR but not the mutant DOR lacking the carboxyl terminus, which contains all three GRK2 phosphorylation sites. DORs with the GRK2 phosphorylation sites modified (M3) or with the acidic residues flanking phosphorylation sites mutated (E355Q/D364N) failed to be phosphorylated in response to agonist stimulation. Agonist-induced GRK2 membrane translocation and GRK-receptor association were observed in cells expressing M3 but not E355Q/D364N. Moreover, over-expression of G beta gamma subunits promoted GRK2 binding to DOR, whereas over-expression of transducin alpha or the carboxyl terminus of GRK2 blocked binding. Further study demonstrated that agonist stimulation induced the formation of a complex containing DOR, GRK2, and G beta gamma subunits in the cell and that agonist-stimulated formation of this complex is essential for the stable localization of GRK2 on the membrane and for its catalytic activity in vivo.

    The Journal of biological chemistry 2003;278;32;30219-26

  • 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

  • The inhibitory gamma subunit of the type 6 retinal cGMP phosphodiesterase functions to link c-Src and G-protein-coupled receptor kinase 2 in a signaling unit that regulates p42/p44 mitogen-activated protein kinase by epidermal growth factor.

    Wan KF, Sambi BS, Tate R, Waters C and Pyne NJ

    Department of Physiology and Pharmacology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 ONR, Scotland, United Kingdom.

    The inhibitory gamma subunit of the retinal photoreceptor type 6 cGMP phosphodiesterase (PDEgamma) is phosphorylated by G-protein-coupled receptor kinase 2 on threonine 62 and regulates the epidermal growth factor- dependent stimulation of p42/p44 mitogen-activated protein kinase in human embryonic kidney 293 cells. We report here that PDEgamma is in a pre-formed complex with c-Src and that stimulation of cells with epidermal growth factor promotes the association of GRK2 with this complex. c-Src has a critical role in the stimulation of the p42/p44 mitogen-activated protein kinase cascade by epidermal growth factor, because c-Src inhibitors block the activation of this kinase by the growth factor. Mutation of Thr-62 (to Ala) in PDEgamma produced a GRK2 phosphorylation-resistant mutant that was less effective in associating with GRK2 in response to epidermal growth factor and did not potentiate the stimulation of p42/p44 mitogen-activated protein kinase by this growth factor. The transcript for a short splice variant version of PDEgamma lacking the Thr-62 phosphorylation site is also expressed in certain mammalian cells and, in common with the Thr-62 mutant, failed to potentiate the stimulatory effect of epidermal growth factor on p42/p44 mitogen-activated protein kinase. The mutation of Thr-22 (to Ala) in PDEgamma, which is a site for phosphorylation by p42/p44 mitogen-activated protein kinase, resulted in a prolonged activation of p42/p44 mitogen-activated protein kinase by epidermal growth factor, suggesting a role for this phosphorylation event in the negative feedback control of PDEgamma.

    The Journal of biological chemistry 2003;278;20;18658-63

  • Pleckstrin homology domain of G protein-coupled receptor kinase-2 binds to PKC and affects the activity of PKC kinase.

    Yang XL, Zhang YL, Lai ZS, Xing FY and Liu YH

    Institute of Gastroenterology, NanFang Hospital, First Military Medical University 510515, Guangzhou, Guangdong Province, China. foru3000@hotmail.com

    Aim: To study the detail mechanism of interaction between PKC and GRK(2) and the effect of GRK(2) on activity of PKC.

    Methods: The cDNA of pleckstrin homology (PH) domain located in GRK(2) residue 548 to 660 was amplified by PCR with the mRNA of human GRK(2) (beta1-adrenergic receptor kinase) as template isolated from human fresh placenta, the expression vector pGEX-PH inserted with the aboved cDNA sequence for GRK(2) PH domain protein and the expression vectors for GST (glutathion-s-transferase) -GRK(2) PH domain fusion protein, BTK (Bruton's tyrosine kinase) PH domain and GST protein were constructed. The expression of GRK(2) in culture mammalian cells (6 cell lines: PC-3, MDCK, SGC7901, Jurkat cell etc.) was determined by SDS-PAGE and Co-immunoprecipitation. The binding of GRK(2) PH domain, GST-GRK(2) PH domain fusion protein and BTK PH domain to PKC in Vitro were detected by SDS-PAGE and Western blot, upon prolonged stimulation of epinephrine, the binding of GRK(2) to PKC was also detected by western blot and Co-immunoprecipitation.

    Results: The binding of GRK(2) PH domain to PKC in Vitro was confirmed by western blot, as were the binding upon prolonged stimulation of epinephrine and the binding of BTK PH domain to PKC. In the present study, GRK(2) PH domain was associated with PKC and down-regulated PKC activity, but Btk PH domain up-regulated PKC activity as compared with GRK(2) PH domain.

    Conclusion: GRK(2) can bind with PKC and down-regulated PKC activity.

    World journal of gastroenterology 2003;9;4;800-3

  • Regulation of melanocortin-4 receptor signaling: agonist-mediated desensitization and internalization.

    Shinyama H, Masuzaki H, Fang H and Flier JS

    Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA.

    Disruption of the hypothalamic melanocortin-4 receptor (MC4R) pathway results in obesity both in humans and rodents, demonstrating a crucial role for hypothalamic MC4Rs in the regulation of energy homeostasis. Because even haploinsufficiency of the MC4R gene can cause obesity in humans and mice, subtle changes in receptor numbers or signaling are likely to impact upon the regulation of food intake and energy expenditure. Little is known about the intracellular regulation of MC4R signaling. Using GT1-7 cells, we show for the first time that the MC4R undergoes ligand-mediated desensitization. We then addressed the possible mechanisms underlying the desensitization using HEK293 and COS-1 cells transfected with hemagglutinin-tagged human MC4R. Preexposure of GT1-7 cells that express endogenous MC4R to the agonist for MC4R, alpha-melanocyte-stimulating hormone, resulted in impaired cAMP formation to a second challenge of alpha-melanocyte-stimulating hormone. The desensitization of MC4R was accompanied by time-dependent internalization of the receptor in HEK293 cells, which was partly inhibited by pretreatment with a specific protein kinase A (PKA) inhibitor, H89. In COS-1 cells, overexpression of dominant-negative G protein-coupled receptor kinase (GRK) 2-K220R partly inhibited the agonist-mediated internalization of MC4R, whereas it did not in HEK293 cells. Overexpression of dominant-negative mutants of beta-arrestin1-V53D and dynamin I-K44A prevented agonist-mediated internalization of MC4R. Mutagenesis studies revealed that Thr312 and Ser329/330 in the C-terminal tail are potential sites for PKA and GRK phosphorylation and may play an essential role in the recruitment of beta-arrestin to the activated receptor. Our data demonstrate that, through PKA-, GRK-, beta-arrestin-, and dynamin-dependent processes, MC4R undergoes internalization in response to agonist, thereby providing novel insights into the regulation of MC4R signaling.

    Endocrinology 2003;144;4;1301-14

  • The amino-terminal domain of G-protein-coupled receptor kinase 2 is a regulatory Gbeta gamma binding site.

    Eichmann T, Lorenz K, Hoffmann M, Brockmann J, Krasel C, Lohse MJ and Quitterer U

    Institut für Pharmakologie und Toxikologie, Versbacher Strasse 9, D-97078 Würzburg, Germany.

    G-protein-coupled receptor kinase 2 (GRK2) is activated by free Gbetagamma subunits. A Gbetagamma binding site of GRK2 is localized in the carboxyl-terminal pleckstrin homology domain. This Gbetagamma binding site of GRK2 also regulates Gbetagamma-stimulated signaling by sequestering free Gbetagamma subunits. We report here that truncation of the carboxyl-terminal Gbetagamma binding site of GRK2 did not abolish the Gbetagamma regulatory activity of GRK2 as determined by the inhibition of a Gbetagamma-stimulated increase in inositol phosphates in cells. This finding suggested the presence of a second Gbetagamma binding site in GRK2. And indeed, the amino terminus of GRK2 (GRK2(1-185)) inhibited a Gbetagamma-stimulated inositol phosphate signal in cells, purified GRK2(1-185) suppressed the Gbetagamma-stimulated phosphorylation of rhodopsin, and GRK2(1-185) bound directly to purified Gbetagamma subunits. The amino-terminal Gbetagamma regulatory site does not overlap with the RGS domain of GRK-2 because GRK2(1-53) with truncated RGS domain inhibited Gbetagamma-mediated signaling with similar potency and efficacy as did GRK2(1-185). In addition to the Gbetagamma regulatory activity, the amino-terminal Gbetagamma binding site of GRK2 affects the kinase activity of GRK2 because antibodies specifically cross-reacting with the amino terminus of GRK2 suppressed the GRK2-dependent phosphorylation of rhodopsin. The antibody-mediated inhibition was released by purified Gbetagamma subunits, strongly suggesting that Gbetagamma binding to the amino terminus of GRK2 enhances the kinase activity toward rhodopsin. Thus, the amino-terminal domain of GRK2 is a previously unrecognized Gbetagamma binding site that regulates GRK2-mediated receptor phosphorylation and inhibits Gbetagamma-stimulated signaling.

    The Journal of biological chemistry 2003;278;10;8052-7

  • Cardiopulmonary bypass decreases G protein-coupled receptor kinase activity and expression in human peripheral blood mononuclear cells.

    Hagen SA, Kondyra AL, Grocott HP, El-Moalem H, Bainbridge D, Mathew JP, Newman MF, Reves JG, Schwinn DA and Kwatra MM

    Pediatric Critical Care Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.

    Background: Cardiopulmonary bypass (CPB) has been implicated in the development of organ injury associated with cardiac surgery. At the molecular level, CPB is accompanied by a pronounced proinflammatory response including an increase in plasma interleukin (IL)-6. The IL-6 has been shown to be increased in rheumatoid arthritis, a chronic inflammatory disease, where it has been implicated in decreasing G protein-coupled receptor kinases (GRKs) in peripheral blood mononuclear cells. Since IL-6 is substantially increased after CPB, the study tested whether the increase of IL-6 during CPB leads to a decrease of GRKs in mononuclear cells. This is important because GRKs regulate the function of G protein-coupled receptors involved in inflammation.

    Methods: Fifteen patients had blood withdrawn before CPB, 2 h after CPB, and on postoperative day one (POD1). Plasma IL-6 concentrations were determined by enzyme-linked immunosorbent assay. The GRK protein expression and activity were determined by Western blot and phosphorylation of rhodopsin using [gamma-(32)P] adenosine triphosphate, respectively.

    Results: Plasma IL-6 increased over 20-fold after CPB and remained increased on POD1. Cytosolic GRK activity in mononuclear cells decreased by 39 +/- 29%; cytosolic GRK2 and membrane-bound GRK6 decreased by 90 +/- 15 and 65 +/- 43%, respectively. The GRK activity and expression of GRK2/GRK6 on POD1 returned to basal levels in many but not all patients.

    Conclusions: The CPB causes a profound decrease in mononuclear cell GRKs, and the recovery of these kinases on POD1 is quite variable. The significance of the variable recovery of GRKs after CPB and their potential role as a marker of clinical outcome deserves further investigation.

    Funded by: NCRR NIH HHS: M01-RR-30; NHLBI NIH HHS: HL57447

    Anesthesiology 2003;98;2;343-8

  • Aberrantly expressed recoverin is functionally associated with G-protein-coupled receptor kinases in cancer cell lines.

    Miyagawa Y, Ohguro H, Odagiri H, Maruyama I, Maeda T, Maeda A, Sasaki M and Nakazawa M

    Department of Ophthalmology, Hirosaki University School of Medicine, 5 Zaifucho Hirosaki 036-8562, Japan.

    Cancer-associated retinopathy (CAR) is an ocular manifestation of a paraneoplastic syndrome whereby immunological reactions toward recoverin (Rec), a retina-specific Ca(2+) binding protein, and its aberrant expression in tumor cells lead to the retinal degeneration. To elucidate functional roles of the aberrantly expression in cancer cells, we performed immunoprecipitation using anti-human Rec mAb. We observed co-precipitation of G-protein-coupled receptor kinases (GRKs) and caveolin-1 with Rec from cell lysates of 293 or SSTW cells. Immunocytochemistry revealed that immunoreactivities toward Rec within the cancer cells were almost identical to those toward GRKs and caveolin-1. The present data strongly suggest that aberrantly expressed Rec should be involved in the GRK-dependent cellular regulation in cancer cells.

    Biochemical and biophysical research communications 2003;300;3;669-73

  • PH domain of G protein-coupled receptor kinase-2 binds to protein kinase C (PKC) and negatively regulates activity of PKC kinase.

    Ji S, Liu X, Li S, Shen L, Li F, Wang J, Han J and Yao L

    Department of Biochemistry and Molecular Biology, The Fourth Military Medical University, Xi'an, Shaaxi, 710032. PR China.

    G protein-coupled receptor kinase-2 (GRK),also known as beta1-adrenergic receptor kinase(beta-ARK1), plays an important role in agonist-induced desensitization of the beta-adrenergic receptors. Activation of protein kinase C (PKC) is able to stimulate phosphorylation and activation of GRKs and induce desensitization of G protein-coupled receptor. However, detail mechanism of interaction between PKC and GRK2 and the effect of GRK2 on activity of PKC remain unknown. Pleckstrin homology (PH) domain is a kind of functionally domain containing about 120 amino acids, which exists on many protein molecules that involve in cellular signal transduction. A PH domain located in GRK2 residue 548 to 660 may play a significant role in mediating interaction between PKC and GRK2. In present study, we revealed that PKC could associate with PH domain of GRK2 in pull-down assay in vitro. Co-immunoprecipitation displayed binding of PKC to GRK2 in intact Jurkat cells after prolonged stimulation of epinephrine. Assay of PKC beta1 kinase activity indicated that the binding of the PH domain of GRK2 to PKC beta 1 could down-regulate activity of PKC beta 1 kinase. Thus, GRK2 may play a negative feedback regulatory role on PKCbeta1 activity in interaction between GRK2 and PKCbeta 1.

    Frontiers in bioscience : a journal and virtual library 2003;8;a34-9

  • Reduction of G protein-coupled receptor kinase 2 expression in U-937 cells attenuates H2 histamine receptor desensitization and induces cell maturation.

    Fernández N, Monczor F, Lemos B, Notcovich C, Baldi A, Davio C and Shayo C

    National Research Council of Argentina, Buenos Aires, Argentina.

    Histamine and H2 agonists transiently induce an important cAMP response in promonocytic U-937 cells but fail to induce monocytic differentiation because of a rapid receptor desensitization mediated by G protein-coupled receptor kinases (GRKs). The aims of the present study were to investigate the participation of GRK2 in the desensitization mechanism of the H2 receptor in U-937 cells by reducing GRK2 levels through antisense technology and to evaluate the differentiating capacity of cells expressing lower GRK2 level, stimulated by H2 agonists. By stable U-937 cell transfection with a GRK2-antisense cDNA, we obtained D5 and A2 cell clones exhibiting a reduction in GRK2 expression and an H3 clone with no significant difference in GRK2 expression from control cells. The cAMP response induced by the H2 agonist in D5 and A2 but not in H3 cells was higher than in U-937 and persisted for a longer period of time, although the number of H2 receptors in D5 and A2 cells was lower than in U-937. Furthermore, D5 and A2 cells treated with H2 agonist showed patterns of c-Fos and CD88 expression consistent with monocytic differentiated cells. Overall, these results indicate a direct correlation between the expression of GRK2 and the desensitization of natively expressed H2 receptors in U-937 cells, suggesting that GRK2 plays a major role in the regulation of these receptors' response. In turn, desensitization process is a key component of H2 receptor signaling, determining the differentiation capability of promonocytic cells.

    Molecular pharmacology 2002;62;6;1506-14

  • Beta 2-adrenergic receptor stimulated, G protein-coupled receptor kinase 2 mediated, phosphorylation of ribosomal protein P2.

    Freeman JL, Gonzalo P, Pitcher JA, Claing A, Lavergne JP, Reboud JP and Lefkowitz RJ

    Howard Hughes Medical Institute and Department of Medicine (Cardiology), Duke University Medical Center, Durham, North Carolina 27710, USA.

    G protein-coupled receptor kinases are well characterized for their ability to phosphorylate and desensitize G protein-coupled receptors (GPCRs). In addition to phosphorylating the beta2-adrenergic receptor (beta2AR) and other receptors, G protein-coupled receptor kinase 2 (GRK2) can also phosphorylate tubulin, a nonreceptor substrate. To identify novel nonreceptor substrates of GRK2, we used two-dimensional gel electrophoresis to find cellular proteins that were phosphorylated upon agonist-stimulation of the beta2AR in a GRK2-dependent manner. The ribosomal protein P2 was identified as an endogenous HEK-293 cell protein whose phosphorylation was increased following agonist stimulation of the beta2AR under conditions where tyrosine kinases, PKC and PKA, were inhibited. P2 along with its other family members, P0 and P1, constitutes a part of the elongation factor-binding site connected to the GTPase center in the 60S ribosomal subunit. Phosphorylation of P2 is known to regulate protein synthesis in vitro. Further, P2 and P1 are shown to be good in vitro substrates for GRK2 with K(M) values approximating 1 microM. The phosphorylation sites in GRK2-phosphorylated P2 are identified (S102 and S105) and are identical to the sites known to regulate P2 activity. When the 60S subunit deprived of endogenous P1 and P2 is reconstituted with GRK2-phosphorylated P2 and unphosphorylated P1, translational activity is greatly enhanced. These findings suggest a previously unrecognized relationship between GPCR activation and the translational control of gene expression mediated by GRK2 activation and P2 phosphorylation and represent a potential novel signaling pathway responsible for P2 phosphorylation in mammals.

    Funded by: NHLBI NIH HHS: HL16037

    Biochemistry 2002;41;42;12850-7

  • Interaction with neuronal calcium sensor NCS-1 mediates desensitization of the D2 dopamine receptor.

    Kabbani N, Negyessy L, Lin R, Goldman-Rakic P and Levenson R

    Department of Pharmacology, Penn State College of Medicine, Hershey, Pennsylvania 17033, USA.

    Dopaminergic transmission within limbic regions of the brain is highly dependent on the regulation of D2 receptor activity. Here we show that the neuronal calcium sensor-1 (NCS-1) can mediate desensitization of D2 dopamine receptors. Analysis of D2 receptors expressed in human embryonic kidney 293 cells indicates that NCS-1 attenuates agonist-induced receptor internalization via a mechanism that involves a reduction in D2 receptor phosphorylation. This effect of NCS-1 was accompanied by an increase in D2 receptor-mediated cAMP inhibition after dopamine stimulation. The ability of NCS-1 to modulate D2 receptor signaling was abolished after a single amino acid mutation in NCS-1 that has been shown to impair the calcium-binding properties of NCS-1. Coimmunoprecipitation experiments from striatal neurons reveal that NCS-1 is found in association with both the D2 receptor and G-protein-coupled receptor kinase 2, a regulator of D2 receptor desensitization. Colocalization of NCS-1 and D2 receptors was examined in both primate and rodent brain. In striatum, NCS-1 and D2 receptors were found to colocalize within sites of synaptic transmission and in close proximity to intracellular calcium stores. NCS-1-D2 receptor interaction may serve to couple dopamine and calcium signaling pathways, thereby providing a critical component in the regulation of dopaminergic signaling in normal and diseased brain.

    Funded by: NIMH NIH HHS: P50-MH44866

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2002;22;19;8476-86

  • Phosphoinositide 3-kinase regulates beta2-adrenergic receptor endocytosis by AP-2 recruitment to the receptor/beta-arrestin complex.

    Naga Prasad SV, Laporte SA, Chamberlain D, Caron MG, Barak L and Rockman HA

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

    Internalization of beta-adrenergic receptors (betaARs) occurs by the sequential binding of beta-arrestin, the clathrin adaptor AP-2, and clathrin. D-3 phosphoinositides, generated by the action of phosphoinositide 3-kinase (PI3K) may regulate the endocytic process; however, the precise molecular mechanism is unknown. Here we demonstrate that betaARKinase1 directly interacts with the PIK domain of PI3K to form a cytosolic complex. Overexpression of the PIK domain displaces endogenous PI3K from betaARK1 and prevents betaARK1-mediated translocation of PI3K to activated beta2ARs. Furthermore, disruption of the betaARK1/PI3K interaction inhibits agonist-stimulated AP-2 adaptor protein recruitment to the beta2AR and receptor endocytosis without affecting the internalization of other clathrin dependent processes such as internalization of the transferrin receptor. In contrast, AP-2 recruitment is enhanced in the presence of D-3 phospholipids, and receptor internalization is blocked in presence of the specific phosphatidylinositol-3,4,5-trisphosphate lipid phosphatase PTEN. These findings provide a molecular mechanism for the agonist-dependent recruitment of PI3K to betaARs, and support a role for the localized generation of D-3 phosphoinositides in regulating the recruitment of the receptor/cargo to clathrin-coated pits.

    Funded by: NHLBI NIH HHS: HL56687, HL61558, R01 HL056687

    The Journal of cell biology 2002;158;3;563-75

  • Orphanin FQ/nociceptin-mediated desensitization of opioid receptor-like 1 receptor and mu opioid receptors involves protein kinase C: a molecular mechanism for heterologous cross-talk.

    Mandyam CD, Thakker DR, Christensen JL and Standifer KM

    Department of Pharmacological and Pharmaceutical Sciences, 521 Building Science and Research 2, University of Houston, Houston, TX 77204-5037, USA.

    Morphine tolerance in vivo is reduced following blockade of the orphanin FQ/nociceptin (OFQ/N)/opioid receptor-like 1 (ORL1) receptor system, suggesting that OFQ/N contributes to the development of morphine tolerance. We previously reported that a 60-min activation of ORL1 receptors natively expressed in BE(2)-C cells desensitized both mu and ORL1 receptor-mediated inhibition of cAMP. Investigating the mechanism(s) of OFQ/N-mediated mu and ORL1 receptor cross-talk, we found that pretreatment with the protein kinase C inhibitor, chelerythrine chloride (1 microM), blocked OFQ/N-mediated homologous desensitization of ORL1 and heterologous desensitization of mu opioid receptors. Furthermore, depletion of PKC by 12-O-tetradecanoylphorbol-13-acetate exposure (48 h, 1 microM) also prevented OFQ/N-mediated mu and ORL1 desensitization. OFQ/N pretreatment resulted in translocation of PKC-alpha, G protein-coupled receptor kinase 2 (GRK2) and GRK3 from the cytosol to the membrane, and this translocation was also blocked by chelerythrine. Reduction of GRK2 and GRK3 levels by antisense, but not sense DNA treatment blocks ORL1 and mu receptor desensitization. This suggests that PKC-alpha is required for GRK2 and GRK3 translocation to the membrane, where GRK can inactivate ORL1 and mu opioid receptors upon rechallenge with the appropriate agonist. Our results demonstrate for the first time the involvement of conventional PKC isozymes in OFQ/N-induced mu-ORL1 cross-talk, and represent a possible mechanism for OFQ/N-induced anti-opioid actions.

    Funded by: NIDA NIH HHS: DA 10738, DA 14171

    The Journal of pharmacology and experimental therapeutics 2002;302;2;502-9

  • Oxidative stress decreases G protein-coupled receptor kinase 2 in lymphocytes via a calpain-dependent mechanism.

    Lombardi MS, Kavelaars A, Penela P, Scholtens EJ, Roccio M, Schmidt RE, Schedlowski M, Mayor F and Heijnen CJ

    Department of Immunology, Laboratory of Psychoneuroimmunology, University Medical Center, Utrecht, The Netherlands.

    G protein-coupled receptor kinase (GRK) 2 plays a crucial role in regulating the extent of desensitization and resensitization of G protein-coupled receptors (GPCRs). We have shown that the expression level of GRK2 in lymphocytes decreases during inflammatory diseases such as arthritis. Reactive oxygen species play an important role in a variety of inflammatory conditions, including arthritis. We demonstrate herein that oxidative stress, induced by exposure of lymphocytes to H(2)O(2), results in a 50% reduction in GRK2 protein levels and GRK activity with no changes in mRNA expression. Treatment of lymphocytes with the tyrosine kinase inhibitor genistein partially reverses the effect of H(2)O(2) on GRK2 levels, although we did not detect direct tyrosine phosphorylation of GRK2. Inhibition of the nonproteasomal protease calpain by calpeptin can prevent the H(2)O(2)-induced GRK2 decrease. In vitro experiments confirm that GRK2 is partially digested by m-calpain in a calcium-dependent way. Functionally, H(2)O(2)-induced decrease in GRK2 levels is associated with an ~70% decrease in agonist-induced beta(2)-adrenergic receptor sequestration. We describe oxidative stress as a novel mechanism for regulation of the intracellular level of GRK2 during inflammatory processes. Moreover, our data demonstrate that oxidative stress may change the functioning of GPCRs via calpain-dependent regulation of GRK2 levels.

    Molecular pharmacology 2002;62;2;379-88

  • Identification of two C-terminal amino acids, Ser(355) and Thr(357), required for short-term homologous desensitization of mu-opioid receptors.

    Wang HL, Chang WT, Hsu CY, Huang PC, Chow YW and Li AH

    Department of Physiology, Chang Gung University School of Medicine, Tao-Yuan, Taiwan, ROC. hlwns@mail.cgu.edu.tw

    Our recent study suggests that a cluster of Ser/Thr residues (T(354)S(355)S(356)T(357)) at the intracellular carboxyl tail of rat mu-opioid receptor (MOR1) is required for the development of short-term homologous desensitization. To investigate the functional role played by individual serine or threonine residue of this (TSST) cluster in the agonist-induced mu-opioid receptor desensitization, point mutant (T354A), (S355A), (S356A) and (T357A) mu-opioid receptors were prepared and stably expressed in human embryonic kidney 293 cells (HEK 293 cells). Similar to wild-type mu-opioid receptors, mutant (T354A) and (S356A) mu-opioid receptors stably expressed in HEK 293 cells developed homologous desensitization after 30 min pretreatment of DAMGO ([D-Ala(2),N-methyl-Phe(4),Gly-ol(5)]enkephalin), a specific mu-opioid receptor agonist. Substituting Ser(355)or Thr(357) with alanine resulted in a significant attenuation of agonist-induced mu-opioid receptor desensitization. In HEK 293 cells stably expressing double mutant (S355A/T357A) mu-opioid receptors, DAMGO pretreatment failed to significantly affect the efficacy and potency by which DAMGO inhibits forskolin-stimulated adenylyl cyclase activity. Consistent with the general belief that agonist-induced phosphorylation of guanine nucleotide binding protein (G protein)-coupled receptors is involved in homologous desensitization. Treating HEK 293 cells expressing wild-type mu-opioid receptors with 5 microM DAMGO for 30 min induced the receptor phosphorylation. Mutation of Ser(355) and Thr(357) also greatly impaired DAMGO-induced mu-opioid receptor phosphorylation. These results suggest that two C-terminal amino acids, Ser(355) and Thr(357), are required for short-term homologous desensitization and agonist-induced phosphorylation of mu-opioid receptors expressed in HEK 293 cells.

    Biochemical pharmacology 2002;64;2;257-66

  • Expression and activity of g protein-coupled receptor kinases in differentiated thyroid carcinoma.

    Métayé T, Menet E, Guilhot J and Kraimps JL

    Biophysics Laboratory, Department of Pathology, Jean Bernard Hospital, BP 577, 86021 Poitiers Cedex, France. t.metaye@chu-poitiers.fr

    Most of the TSH effects on the proliferation and differentiation of thyroid cells are mediated by cAMP via an adenylyl cyclase-activating Gs protein. TSH receptor responsiveness in cell cultures, is regulated by G protein-coupled receptor kinase (GRK) 2 and 5. To determine whether an alteration in activity and expression of GRKs might be associated with variable levels of TSH receptor desensitization in vivo, we studied human thyroid tissues including 21 normal tissues and 18 differentiated carcinomas. GRK activity was assayed by rhodopsin phosphorylation, and GRK protein and mRNA expressions assessed by immunoblotting and real-time quantitative RT-PCR, respectively. GRK2 and GRK5 were found as the predominant isoforms in the human thyroid. GRK5 protein expression was significantly decreased in differentiated thyroid carcinoma (P < 0.02) and paralleled a decrease in GRK mRNA expression (P < 0.02). In contrast, no difference in protein and mRNA levels of GRK2 were observed between normal and cancerous thyroid tissues. Although GRK2 protein levels correlated with GRK activities, we demonstrated a significant increase in GRK activity in differentiated thyroid carcinoma (P < 0.02). Less TSH receptor desensitization occurred in differentiated carcinoma than in normal thyroid tissue, as judged by TSH-stimulated cAMP response in human thyroid cells in primary culture. In conclusion, this study indicates that GRK2 activity and GRK5 expression have opposite regulations in cancer cells. Furthermore, the decrease in GRK5 expression may underlie the reduction in homologous desensitization of the TSH receptor in differentiated thyroid carcinoma, contributing to explain the increased cAMP levels in these tumors.

    The Journal of clinical endocrinology and metabolism 2002;87;7;3279-86

  • Determination of bradykinin B2 receptor in vivo phosphorylation sites and their role in receptor function.

    Blaukat A, Pizard A, Breit A, Wernstedt C, Alhenc-Gelas F, Muller-Esterl W and Dikic I

    Ludwig Institute for Cancer Research, Box 595, S-75124 Uppsala, Sweden.

    Reversible phosphorylation plays important roles in G protein-coupled receptor signaling, desensitization, and endocytosis, yet the precise location and role of in vivo phosphorylation sites is unknown for most receptors. Using metabolic 32P labeling and phosphopeptide sequencing we provide a complete phosphorylation map of the human bradykinin B2 receptor in its native cellular environment. We identified three serine residues, Ser(339), Ser(346), and Ser(348), at the C-terminal tail as principal phosphorylation sites. Constitutive phosphorylation occurs at Ser(348), while ligand-induced phosphorylation is found at Ser(339) and Ser(346)/Ser(348) that could be executed by several G protein-coupled receptor kinases. In addition, we found a protein kinase C-dependent phosphorylation of Ser(346) that was mutually exclusive with the basal phosphorylation at Ser(348) and therefore may be implicated in differential regulation of B2 receptor activation. Functional analysis of receptor mutants revealed that a low phosphorylation stoichiometry is sufficient to initiate receptor sequestration while a clustered phosphorylation around Ser(346) is necessary for desensitization of the B2 receptor-induced phospholipase C activation. This was further supported by the specifically reduced Ser(346)/Ser(348) phosphorylation observed upon stimulation with a nondesensitizing B2 receptor agonist. The differential usage of clustered phosphoacceptor sites points to distinct roles of multiple kinases in controlling G protein-coupled receptor function.

    The Journal of biological chemistry 2001;276;44;40431-40

  • G-protein coupled receptor kinase 2 and 3 expression in human detrusor cultured smooth muscle cells.

    Obara K, Arai K, Tomita Y, Hatano A and Takahashi K

    Department of Regenerative and Transplant Medicine, Graduate School of Medical and Dental Sciences, Niigata University, Japan. obarak@med.niigata-u.ac.jp

    The aim of this study was to investigate the expression of G-protein coupled receptor kinases (GRKs) mRNA by RT-PCR and GRKs protein by immunohistochemistry in human detrusor cultured smooth muscle. Primary cultures of human detrusor smooth muscle cells were established using the explant method from three normal bladders. The expression of each GRK, beta-adrenergic receptor and muscarinic acethylcholine receptor (mAchR) mRNA was examined by reverse transcription polymerase chain reaction (RT-PCR). Immunohistochemical staining was also performed using primary antibodies for GRKs. The GRK2 and GRK3 transcripts were detected by RT-PCR. The m2, m3 and m5 mAchR and beta1, beta2 and beta3 adrenergic receptor subtypes mRNA were also detected. Using immunohistochemistry, both GRK2 and GRK3 were found to be expressed in detrusor smooth muscle cells. These results demonstrated the existence of GRK2 and GRK3 and the co-expression of m2, m3 and m5 mAchR and beta1, beta2 and beta3 adrenergic receptor subtypes in detrusor smooth muscle cells. The possibility exists that these kinases play a role in the desensitization mechanism of mAchR and beta adrenergic receptors.

    Urological research 2001;29;5;325-9

  • The scaffold protein gravin (cAMP-dependent protein kinase-anchoring protein 250) binds the beta 2-adrenergic receptor via the receptor cytoplasmic Arg-329 to Leu-413 domain and provides a mobile scaffold during desensitization.

    Fan G, Shumay E, Wang H and Malbon CC

    Department of Molecular Pharmacology, Diabetes & Metabolic Diseases Research Program, University Medical Center, SUNY/Stony Brook, Stony Brook, New York 11794-8651, USA.

    The cyclic AMP-dependent kinase-anchoring proteins (AKAPs) function as scaffolds for a wide-range of protein-protein interactions. The 250-kDa AKAP known as gravin plays a central role in organizing G-protein-coupled receptors to the protein kinases and phosphatases that regulate receptor function in desensitization, resensitization, and sequestration. Although gravin is critical for G-protein-linked receptor biology, the molecular features of the receptor necessary for interaction with this scaffold are not known. Herein, we map the regions of the beta(2)-adrenergic receptor that are required for binding to gravin. Intracellular loops 1, 2, and 3 appear not to participate in the binding of the receptor to the scaffold. In contrast, the C-terminal cytoplasmic region of the receptor (Arg-329 to Leu-413) competes readily for the binding of the beta(2)-adrenergic receptor by gravin, both using in vitro and in vivo assays. C-terminally truncated peptides with sequences ranging from Arg-329 to Leu-342 (13 aminoacyl residues), to Asn-352 (23 residues), to Tyr-366 (37 residues), to Asp-380 (51 residues), or to His-390 (61 residues), as well as N-terminally truncated peptides from Gln-391 to Leu-413 (23 residues) or Leu-381 to Leu-413 (33 residues) displayed no ability to block binding of receptor to gravin. The combination of Arg-329 to His-390 peptide and Gln-391 to Leu-413 peptide, however, reconstitutes a fragmented but full-length C-terminal region and also potently blocks the ability of gravin to bind the beta(2)-adrenergic receptor. The gravin-receptor interaction was examined in response to agonist by confocal microscopy. Remarkably, the association of the receptor with gravin was not disrupted during agonist-induced sequestration. The receptor-scaffold complex was maintained during agonist-induced sequestration. These data, in agreement with the biochemical data, reveal that gravin binds the receptor through the beta(2)-adrenergic receptor C-terminal cytoplasmic domain and that this interaction is maintained as the receptor is internalized. This is the first report of an AKAP scaffold protein translocating with its receptor, in this case a G-protein-coupled receptor.

    The Journal of biological chemistry 2001;276;26;24005-14

  • Regulation of membrane targeting of the G protein-coupled receptor kinase 2 by protein kinase A and its anchoring protein AKAP79.

    Cong M, Perry SJ, Lin FT, Fraser ID, Hu LA, Chen W, Pitcher JA, Scott JD and Lefkowitz RJ

    Howard Hughes Medical Institute, Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.

    The beta2 adrenergic receptor (beta2AR) undergoes desensitization by a process involving its phosphorylation by both protein kinase A (PKA) and G protein-coupled receptor kinases (GRKs). The protein kinase A-anchoring protein AKAP79 influences beta2AR phosphorylation by complexing PKA with the receptor at the membrane. Here we show that AKAP79 also regulates the ability of GRK2 to phosphorylate agonist-occupied receptors. In human embryonic kidney 293 cells, overexpression of AKAP79 enhances agonist-induced phosphorylation of both the beta2AR and a mutant of the receptor that cannot be phosphorylated by PKA (beta2AR/PKA-). Mutants of AKAP79 that do not bind PKA or target to the beta2AR markedly inhibit phosphorylation of beta2AR/PKA-. We show that PKA directly phosphorylates GRK2 on serine 685. This modification increases Gbetagamma subunit binding to GRK2 and thus enhances the ability of the kinase to translocate to the membrane and phosphorylate the receptor. Abrogation of the phosphorylation of serine 685 on GRK2 by mutagenesis (S685A) or by expression of a dominant negative AKAP79 mutant reduces GRK2-mediated translocation to beta2AR and phosphorylation of agonist-occupied beta2AR, thus reducing subsequent receptor internalization. Agonist-stimulated PKA-mediated phosphorylation of GRK2 may represent a mechanism for enhancing receptor phosphorylation and desensitization.

    Funded by: NHLBI NIH HHS: HL16037; NIGMS NIH HHS: GM48231

    The Journal of biological chemistry 2001;276;18;15192-9

  • Phosphorylation of GRK2 by protein kinase C abolishes its inhibition by calmodulin.

    Krasel C, Dammeier S, Winstel R, Brockmann J, Mischak H and Lohse MJ

    Institut für Pharmakologie und Toxikologie, Versbacher Str. 9, D-97078 Würzburg, Germany. krasel@wpxx02.toxi.uni-weirzburg.de

    G-protein-coupled receptor kinases (GRKs) are important regulators of G-protein-coupled receptor function. Two members of this family L, GRK2 and GRK5 L, have been shown to be substrates for protein kinase C (PKC). Whereas PKC-mediated phosphorylation results in inhibition of GRK5, it increases the activity of GRK2 toward its substrates probably through increased affinity for receptor-containing membranes. We show here that this increase in activity may be caused by relieving a tonic inhibition of GRK2 by calmodulin. In vitro, GRK2 was preferentially phosphorylated by PKC isoforms alpha, gamma, and delta. Two-dimensional peptide mapping of PKCalpha-phosphorylated GRK2 showed a single site of phosphorylation, which was identified as serine 29 by HPLC-MS. A S29A mutant of GRK2 was not phosphorylated by PKC in vitro and showed no phorbol ester-stimulated phosphorylation when transfected into human embryonic kidney (HEK)293 cells. Serine 29 is located in the calmodulin-binding region of GRK2, and binding of calmodulin to GRK2 results in inhibition of kinase activity. This inhibition was almost completely abolished in vitro when GRK2 was phosphorylated by PKC. These data suggest that calmodulin may be an inhibitor of GRK2 whose effects can be abolished with PKC-mediated phosphorylation of GRK2.

    The Journal of biological chemistry 2001;276;3;1911-5

  • Identification of G protein-coupled receptor kinase 2 phosphorylation sites responsible for agonist-stimulated delta-opioid receptor phosphorylation.

    Guo J, Wu Y, Zhang W, Zhao J, Devi LA, Pei G and Ma L

    National Laboratory of Medical Neurobiology, Fudan University Medical Center, Shanghai, People's Republic of China.

    Agonist-induced receptor phosphorylation is an initial step in opioid receptor desensitization, a molecular mechanism of opioid tolerance and dependence. Our previous research suggested that agonist-induced delta-opioid receptor (DOR) phosphorylation occurs at the receptor carboxyl terminal domain. The current study was carried out to identify the site of DOR phosphorylation during agonist stimulation and the kinases catalyzing this reaction. Truncation (Delta15) or substitutions (T358A, T361A, and S363G single or triple mutants) at the DOR cytoplasmic tail caused 80 to 100% loss of opioid-stimulated receptor phosphorylation, indicating that T358, T361, and S363 all contribute and are cooperatively involved in agonist-stimulated DOR phosphorylation. Coexpression of GRK2 strongly enhanced agonist-stimulated phosphorylation of the wild-type DOR (WT), but Delta15 or mutant DOR (T358A/T361A/S363G) failed to show any detectable phosphorylation under these conditions. These results demonstrate that T358, T361, and S363 are required for agonist-induced and GRK-mediated receptor phosphorylation. Agonist-induced receptor phosphorylation was severely impaired by substitution of either T358 or S363 with aspartic acid residue, but phosphorylation of the T361D mutant was comparable with that of WT. In the presence of exogenously expressed GRK2, phosphorylation levels of T358D and S363D mutants were approximately half of that of WT, whereas significant phosphorylation of the T358/S363 double-point mutant was not detected. These results indicate that both T358 and S363 residues at the DOR carboxyl terminus are capable of serving cooperatively as phosphate acceptor sites of GRK2 in vivo. Taken together, we have demonstrated that agonist-induced opioid receptor phosphorylation occurs exclusively at two phosphate acceptor sites (T358 and S363) of GRK2 at the DOR carboxyl terminus. These results represent the identification of the GRK phosphorylation site on an opioid receptor for the first time and demonstrate that GRK is the prominent kinase responsible for agonist-induced opioid receptor phosphorylation in vivo.

    Molecular pharmacology 2000;58;5;1050-6

  • Phosphorylation of phosducin and phosducin-like protein by G protein-coupled receptor kinase 2.

    Ruiz-Gómez A, Humrich J, Murga C, Quitterer U, Lohse MJ and Mayor F

    Departamento de Biología Molecular and Centro de Biologia Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientificas, Universidad Autónoma de Madrid, E-28049 Madrid, Spain.

    G protein-coupled receptor kinase 2 (GRK2) is able to phosphorylate a variety of agonist-occupied G protein-coupled receptors (GPCR) and plays an important role in GPCR modulation. However, recent studies suggest additional cellular functions for GRK2. Phosducin and phosducin-like protein (PhLP) are cytosolic proteins that bind Gbetagamma subunits and act as regulators of G-protein signaling. In this report, we identify phosducin and PhLP as novel GRK2 substrates. The phosphorylation of purified phosducin and PhLP by recombinant GRK2 proceeds rapidly and stoichiometrically (0.82 +/- 0.1 and 0.83 +/- 0.09 mol of P(i)/mol of protein, respectively). The phosphorylation reactions exhibit apparent K(m) values in the range of 40-100 nm, strongly suggesting that both proteins could be endogenous targets for GRK2 activity. Our data show that the site of phosducin phosphorylation by GRK2 is different and independent from that previously reported for the cAMP-dependent protein kinase. Analysis of GRK2 phosphorylation of a variety of deletion mutants of phosducin and PhLP indicates that the critical region for GRK2 phosphorylation is localized in the C-terminal domain of both phosducin and PhLP (between residues 204 and 245 and 195 and 218, respectively). This region is important for the interaction of these proteins with G beta gamma subunits. Phosphorylation of phosducin by GRK2 markedly reduces its G beta gamma binding ability, suggesting that GRK2 may modulate the activity of the phosducin protein family by disrupting this interaction. The identification of phosducin and PhLP as new substrates for GRK2 further expands the cellular roles of this kinase and suggests new mechanisms for modulating GPCR signal transduction.

    The Journal of biological chemistry 2000;275;38;29724-30

  • Synucleins are a novel class of substrates for G protein-coupled receptor kinases.

    Pronin AN, Morris AJ, Surguchov A and Benovic JL

    Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.

    G protein-coupled receptor kinases (GRKs) specifically recognize and phosphorylate the agonist-occupied form of numerous G protein-coupled receptors (GPCRs), ultimately resulting in desensitization of receptor signaling. Until recently, GPCRs were considered to be the only natural substrates for GRKs. However, the recent discovery that GRKs also phosphorylate tubulin raised the possibility that additional GRK substrates exist and that the cellular role of GRKs may be much broader than just GPCR regulation. Here we report that synucleins are a novel class of GRK substrates. Synucleins (alpha, beta, gamma, and synoretin) are 14-kDa proteins that are highly expressed in brain but also found in numerous other tissues. alpha-Synuclein has been linked to the development of Alzheimer's and Parkinson's diseases. We found that all synucleins are GRK substrates, with GRK2 preferentially phosphorylating the alpha and beta isoforms, whereas GRK5 prefers alpha-synuclein as a substrate. GRK-mediated phosphorylation of synuclein is activated by factors that stimulate receptor phosphorylation, such as lipids (all GRKs) and Gbetagamma subunits (GRK2/3), suggesting that GPCR activation may regulate synuclein phosphorylation. GRKs phosphorylate synucleins at a single serine residue within the C-terminal domain. Although the function of synucleins remains largely unknown, recent studies have demonstrated that these proteins can interact with phospholipids and are potent inhibitors of phospholipase D2 (PLD2) in vitro. PLD2 regulates the breakdown of phosphatidylcholine and has been implicated in vesicular trafficking. We found that GRK-mediated phosphorylation inhibits synuclein's interaction with both phospholipids and PLD2. These findings suggest that GPCRs may be able to indirectly stimulate PLD2 activity via their ability to regulate GRK-promoted phosphorylation of synuclein.

    Funded by: NIGMS NIH HHS: GM44944

    The Journal of biological chemistry 2000;275;34;26515-22

  • The GIT family of ADP-ribosylation factor GTPase-activating proteins. Functional diversity of GIT2 through alternative splicing.

    Premont RT, Claing A, Vitale N, Perry SJ and Lefkowitz RJ

    Departments of Medicine and Biochemistry, Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710, USA. richard.premont@duke.edu

    We recently characterized a novel protein, GIT1, that interacts with G protein-coupled receptor kinases and possesses ADP-ribosylation factor (ARF) GTPase-activating protein activity. A second ubiquitously expressed member of the GIT protein family, GIT2, has been identified in data base searches. GIT2 undergoes extensive alternative splicing and exists in at least 10 and potentially as many as 33 distinct forms. The longest form of GIT2 is colinear with GIT1 and shares the same domain structure, whereas one major splice variant prominent in immune tissues completely lacks the carboxyl-terminal domain. The other 32 potential variants arise from the independent alternative splicing of five internal regions in the center of the molecule but share both the amino-terminal ARF GTPase-activating protein domain and carboxyl-terminal domain. Both the long and short carboxyl-terminal variants of GIT2 are active as GTPase-activating proteins for ARF1, and both also interact with G protein-coupled receptor kinase 2 and with p21-activated kinase-interacting exchange factors complexed with p21-activated kinase but not with paxillin. Cellular overexpression of the longest variant of GIT2 leads to inhibition of beta(2)-adrenergic receptor sequestration, whereas the shortest splice variant appears inactive. Although GIT2 shares many properties with GIT1, it also exhibits both structural and functional diversity due to tissue-specific alternative splicing.

    Funded by: NHLBI NIH HHS: HL16037

    The Journal of biological chemistry 2000;275;29;22373-80

  • Gravin-mediated formation of signaling complexes in beta 2-adrenergic receptor desensitization and resensitization.

    Lin F, Wang Hy and Malbon CC

    Department of Molecular Pharmacology, Diabetes and Metabolic Diseases Research Program, University Medical Center, State University of New York, Stony Brook, New York 11794-8651, USA.

    Agonist-induced desensitization and resensitization of G-protein-linked receptors involve the interaction of receptors with protein kinases, phosphatases, beta-arrestin, and clathrin organized by at least one scaffold protein. The dynamic composition of the signaling complexes and the role of the scaffold protein AKAP250 (gravin) in agonist-induced attenuation and recovery of beta-adrenergic receptors were explored by co-immunoprecipitation of target elements, antisense suppression, and confocal microscopy. Gravin associated with unstimulated receptor, and the association was increased significantly after agonist stimulation for up to 60 min. Agonist stimulation also induced a robust association of the receptor-gravin complex with protein kinases A and C, G-protein-linked receptor kinase-2, beta-arrestin, and clathrin. Confocal microscopy of the green fluorescence protein-tagged beta(2)-adrenergic receptor showed that the receptor underwent sequestration after agonist stimulation. Suppression of gravin expression via antisense oligodeoxynucleotides disrupted agonist-induced association of the receptor with G-protein-linked receptor kinase-2, beta-arrestin, and clathrin as well as receptor recovery from desensitization. Gravin deficiency also inhibited agonist-induced sequestration. These data reveal that gravin-mediated formation of signaling complexes with protein kinases/phosphatases, beta-arrestin, and clathrin is essential in agonist-induced internalization and resensitization of G-protein-linked receptors.

    The Journal of biological chemistry 2000;275;25;19025-34

  • G protein-coupled receptor kinase-5 regulates thrombin-activated signaling in endothelial cells.

    Tiruppathi C, Yan W, Sandoval R, Naqvi T, Pronin AN, Benovic JL and Malik AB

    Department of Pharmacology, College of Medicine, University of Illinois, Chicago, IL 60612, USA. tiruc@uic.edu

    We studied the function of G protein-coupled receptor kinases (GRKs) in the regulation of thrombin-activated signaling in endothelial cells. GRK2, GRK5, and GRK6 isoforms were expressed predominantly in endothelial cells. The function of these isoforms was studied by expressing wild-type and dominant negative (dn) mutants in endothelial cells. We determined the responses to thrombin, which activates intracellular signaling in endothelial cells by cleaving the NH(2) terminus of the G protein-coupled proteinase-activated receptor-1 (PAR-1). We measured changes in phosphoinositide hydrolysis and intracellular Ca(2+) concentration ([Ca(2+)](i)) in response to thrombin as well as the state of endothelial activation. In the latter studies, the transendothelial monolayer electrical resistance, a measure of the loss of endothelial barrier function, was measured in real time. Of the three isoforms, GRK5 overexpression was selective in markedly reducing the thrombin-activated phosphoinositide hydrolysis and increased [Ca(2+)](i). GRK5 overexpression also inhibited the thrombin-induced decrease in endothelial monolayer resistance by 75%. These effects of GRK5 overexpression occurred in association with the specific increase in the thrombin-induced phosphorylation of PAR-1. In contrast to the effects of GRK5 overexpression, the expression of the dn-GRK5 mutant produced a long-lived increase in [Ca(2+)](i) in response to thrombin, whereas dn-GRK2 had no effect. These results indicate the crucial role of the GRK5 isoform in the mechanism of thrombin-induced desensitization of PAR-1 in endothelial cells.

    Funded by: NHLBI NIH HHS: HL45638, R01 HL045638; NIGMS NIH HHS: GM44944, GM58531, R01 GM044944, R01 GM058531

    Proceedings of the National Academy of Sciences of the United States of America 2000;97;13;7440-5

  • Human NK cells express CC chemokine receptors 4 and 8 and respond to thymus and activation-regulated chemokine, macrophage-derived chemokine, and I-309.

    Inngjerdingen M, Damaj B and Maghazachi AA

    Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.

    NK cells respond to various chemokines, suggesting that they express receptors for these chemokines. In this paper, we show that IL-2-activated NK (IANK) cells express CC chemokine receptor 4 (CCR4) and CCR8, as determined by flow cytometric, immunoblot, and RNase protection assays. Macrophage-derived chemokine (MDC), the ligand for CCR4, induces the phosphorylation of CCR4 within 0.5 min of activating IANK cells with this ligand. This is corroborated with the recruitment of G protein-coupled receptor kinases 2 and 3 and their association with CCR4 in IANK cell membranes. Also, CCR4 is internalized between 5 and 45 min but reappears in the membranes after 60 min of stimulation with MDC. MDC, thymus and activation-regulated chemokine (TARC), and I-309 induce the chemotaxis of IANK cells, an activity that is inhibited upon pretreatment of these cells with pertussis toxin, suggesting that receptors for these chemokines are coupled to pertussis toxin-sensitive G proteins. In the calcium release assay, cross-desensitization experiments showed that TARC completely desensitizes the calcium flux response induced by MDC or I-309, whereas both MDC and I-309 partially desensitize the calcium flux response induced by TARC. These results suggest that TARC utilizes CCR4 and CCR8. Our results are the first to show that IL-2-activated NK cells express CCR4 and CCR8, suggesting that these receptors are not exclusive for Th2 cells.

    Journal of immunology (Baltimore, Md. : 1950) 2000;164;8;4048-54

  • Mutational analysis of Gbetagamma and phospholipid interaction with G protein-coupled receptor kinase 2.

    Carman CV, Barak LS, Chen C, Liu-Chen LY, Onorato JJ, Kennedy SP, Caron MG and Benovic JL

    Department of Microbiology & Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.

    Agonist-dependent regulation of G protein-coupled receptors is dependent on their phosphorylation by G protein-coupled receptor kinases (GRKs). GRK2 and GRK3 are selectively regulated in vitro by free Gbetagamma subunits and negatively charged membrane phospholipids through their pleckstrin homology (PH) domains. However, the molecular binding determinants and physiological role for these ligands remain unclear. To address these issues, we generated an array of site-directed mutants within the GRK2 PH domain and characterized their interaction with Gbetagamma and phospholipids in vitro. Mutation of several residues in the loop 1 region of the PH domain, including Lys-567, Trp-576, Arg-578, and Arg-579, resulted in a loss of receptor phosphorylation, likely via disruption of phospholipid binding, that was reversed by Gbetagamma. Alternatively, mutation of residues distal to the C-terminal amphipathic alpha-helix, including Lys-663, Lys-665, Lys-667, and Arg-669, resulted in decreased responsiveness to Gbetagamma. Interestingly, mutation of Arg-587 in beta-sheet 3, a region not previously thought to interact with Gbetagamma, resulted in a specific and profound loss of Gbetagamma responsiveness. To further characterize these effects, two mutants (GRK2(K567E/R578E) and GRK2(R587Q)) were expressed in Sf9 cells and purified. Analysis of these mutants revealed that GRK2(K567E/R578E) was refractory to stimulation by negatively charged phospholipids but bound Gbetagamma similar to wild-type GRK2. In contrast, GRK2(R587Q) was stimulated by acidic phospholipids but failed to bind Gbetagamma. In order to examine the role of phospholipid and Gbetagamma interaction in cells, wild-type and mutant GRK2s were expressed with a beta(2)-adrenergic receptor (beta(2)AR) mutant that is responsive to GRK2 phosphorylation (beta(2)AR(Y326A)). In these cells, GRK2(K567E/R578E) and GRK2(R587Q) were largely defective in promoting agonist-dependent phosphorylation and internalization of beta(2)AR(Y326A). Similarly, wild-type GRK2 but not GRK2(K567E/R578E) or GRK2(R587Q) promoted morphinedependent phosphorylation of the mu-opioid receptor in cells. Thus, we have (i) identified several specific GRK2 binding determinants for Gbetagamma and phospholipids, and (ii) demonstrated that Gbetagamma binding is the limiting step for GRK2-dependent receptor phosphorylation in cells.

    Funded by: NCI NIH HHS: 5-T32-CA09662; NIGMS NIH HHS: GM44944

    The Journal of biological chemistry 2000;275;14;10443-52

  • Agonist-dependent modulation of G protein-coupled receptor kinase 2 by mitogen-activated protein kinases.

    Elorza A, Sarnago S and Mayor F

    Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Universidad Autónoma, Madrid, Spain.

    A variety of G protein-coupled receptors (GPCRs) are phosphorylated by G protein-coupled receptor kinase 2 (GRK2). This event promotes the binding of regulatory proteins termed beta-arrestins to GPCRs, leading to uncoupling from G proteins and receptor internalization. Recent data indicate that GRK2 and beta-arrestins also play an important role in the stimulation of the extracellular signal-regulated kinases (ERK)/mitogen-activated protein kinase (MAPK) cascade by GPCRs. In this report, we have investigated the existence of functional interactions between GRK2 and MAPK. We show that activation of beta(2)-adrenergic receptors (beta(2)-AR) promotes the rapid association of GRK2 and MAPK in living cells, as assessed by coimmunoprecipitation experiments in COS-7 cells transfected with beta(2)-AR, GRK2, and an epitope-tagged MAPK. Coimmunoprecipitation of MAPK and GRK2 is blocked by inhibition of the MAPK cascade and is not observed upon activation of MAPK in the absence of beta(2)-AR stimulation, thus indicating that both an active MAPK and agonist occupancy of GPCR are required for the association to occur. Interestingly, we have found that purified ERK1/MAPK can directly phosphorylate the C-terminal domain of GRK2, and that the phosphorylation process is favored by the presence of Gbetagamma-subunits or an activated receptor. Furthermore, GRK2 phosphorylation by MAPK leads to a decreased activity of GRK2 toward GPCR. Taken together, our results suggest that stimulation of GPCRs promotes the rapid association of GRK2 and MAPK leading to modulation of GRK2 functionality, thus putting forward a new feedback mechanism for the regulation of GPCR signaling.

    Molecular pharmacology 2000;57;4;778-83

  • Interaction between the conserved region in the C-terminal domain of GRK2 and rhodopsin is necessary for GRK2 to catalyze receptor phosphorylation.

    Gan XQ, Wang JY, Yang QH, Li Z, Liu F, Pei G and Li L

    Shanghai Institute of Biochemistry, Shanghai 200031, People's Republic of China.

    The C-terminal domain of G protein-coupled receptor kinases (GRKs) consists of a conserved region and a variable region, and the variable region has been shown to direct the membrane translocation of cytosolic enzymes. The present work has revealed that the C-terminal domain may also be involved in kinase-receptor interaction that is primarily mediated by the conserved region. Truncation of the C-terminal domain or deletion of the conserved region in this domain of GRK2 resulted in a complete loss of its ability to phosphorylate rhodopsin and in an obvious decrease in its sensitivity to receptor-mediated phosphorylation of a peptide substrate. On the contrary, deletion of the betagamma subunit binding region in the C-terminal domain of GRK2 did not significantly alter the ability of the enzyme to phosphorylate rhodopsin. In addition, the recombinant proteins that represent the C-terminal domain and the conserved region of GRK2 could inhibit GRK2-mediated phosphorylation of rhodopsin and receptor-mediated activation of GRK2 but not GRK2-mediated phosphorylation of the peptide substrate. Furthermore, the conserved region as well as the C-terminal domain could directly bind rhodopsin in vitro. These results indicate that the C-terminal domain, or more precisely, the conserved region of this domain, is important for enzyme-receptor interaction and that this interaction is required for GRK2 to catalyze receptor phosphorylation.

    The Journal of biological chemistry 2000;275;12;8469-74

  • Constitutive phosphorylation of the Parkinson's disease associated alpha-synuclein.

    Okochi M, Walter J, Koyama A, Nakajo S, Baba M, Iwatsubo T, Meijer L, Kahle PJ and Haass C

    Adolf-Butenandt Institute, Department of Biochemistry, Ludwig-Maximilians University, 80336 Munich, Germany.

    alpha-Synuclein has been implicated in the pathogenesis of Parkinson's disease, since rare autosomal dominant mutations are associated with early onset of the disease and alpha-synuclein was found to be a major constituent of Lewy bodies. We have analyzed alpha-synuclein expression in transfected cell lines. In pulse-chase experiments alpha-synuclein appeared to be stable over long periods (t((1)/(2)) 54 h) and no endoproteolytic processing was observed. alpha-Synuclein was constitutively phosphorylated in human kidney 293 cells as well as in rat pheochromocytoma PC12 cells. In both cell lines phosphorylation was highly sensitive to phosphatases, since okadaic acid markedly stabilized phosphate incorporation. Phosphoamino acid analysis revealed that phosphorylation occurred predominantly on serine. Using site-directed mutagenesis we have identified a major phosphorylation site at serine 129 within the C-terminal domain of alpha-synuclein. An additional site, which was phosphorylated less efficiently, was mapped to serine 87. The major phosphorylation site was located within a consensus recognition sequence of casein kinase 1 (CK-1). In vitro experiments and two-dimensional phosphopeptide mapping provided further evidence that serine 129 was phosphorylated by CK-1 and CK-2. Moreover, phosphorylation of serine 129 was reduced in vivo upon inhibition of CK-1 or CK-2. These data demonstrate that alpha-synuclein is constitutively phosphorylated within its C terminus and may indicate that the function of alpha-synuclein is regulated by phosphorylation/dephosphorylation.

    The Journal of biological chemistry 2000;275;1;390-7

  • Feedback inhibition of G protein-coupled receptor kinase 2 (GRK2) activity by extracellular signal-regulated kinases.

    Pitcher JA, Tesmer JJ, Freeman JL, Capel WD, Stone WC and Lefkowitz RJ

    Howard Hughes Medical Institute, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.

    G protein-coupled receptor kinase (GRK)-mediated receptor phosphorylation and beta-arrestin binding uncouple G protein-coupled receptors (GPCRs) from their respective G proteins and initiates the process of receptor internalization. In the case of the beta(2)-adrenergic receptor and lysophosphatidic acid receptor, these processes can lead to ERK activation. Here we identify a novel mechanism whereby the activity of GRK2 is regulated by feedback inhibition. GRK2 is demonstrated to be a phosphoprotein in cells. Mass spectrometry and mutational analysis localize the site of phosphorylation on GRK2 to a carboxyl-terminal serine residue (Ser(670)). Phosphorylation at Ser(670) impairs the ability of GRK2 to phosphorylate both soluble and membrane-incorporated receptor substrates and dramatically attenuates Gbetagamma-mediated activation of this enzyme. Ser(670) is located in a peptide sequence that conforms to an ERK consensus phosphorylation sequence, and in vitro, in the presence of heparin, ERK1 phosphorylates GRK2. Inhibition of ERK activity in HEK293 cells potentiates GRK2 activity, whereas, conversely, ERK activation inhibits GRK2 activity. The discovery that ERK phosphorylates and inactivates GRK2 suggests that ERK participates in a feedback regulatory loop. By negatively regulating GRK-mediated receptor phosphorylation, beta-arrestin-mediated processes such as Src recruitment and clathrin-mediated internalization, which are required for GPCR-mediated ERK activation, are inhibited, thus dampening further ERK activation.

    Funded by: NHLBI NIH HHS: HL16037

    The Journal of biological chemistry 1999;274;49;34531-4

  • Expression of G-protein-coupled receptor kinases in pregnant term and non-pregnant human myometrium.

    Brenninkmeijer CB, Price SA, López Bernal A and Phaneuf S

    University of Oxford, Nuffield Department of Obstetrics and Gynaecology, Women's Centre, Oxford OX3 9DU, UK.

    There is evidence for hormonal receptor desensitisation in human myometrium, but little is known about the mechanisms involved in the loss of myometrial response to agonists such as beta(2)-adrenergic agonists, prostaglandin gamma and oxytocin. It is well known that the receptors for these hormones are coupled to G-proteins. The first step of receptor desensitisation is the phosphorylation of activated receptors by a G-protein-coupled receptor kinase (GRK). GRKs are members of a multigene family and the various subtypes differ in their localisation, regulation and mode of action. We have used Western blotting and reverse transcription PCR to identify the GRKs present in human myometrium from pregnant and non-pregnant women as well as in cultured human myometrial cells. We have found that human myometrium expresses the GRK subtypes 2, 4gamma, 5 and 6. On the other hand, GRK3 and the isoforms GRK4alpha, beta and delta were not found in myometrial tissue. Our data indicate that GRK2 is only expressed in pregnant term myometrium and is not found in non-pregnant tissue. Moreover, GRK6 appears to be expressed at a much higher level in pregnant term tissue than in non-pregnant myometrium. Our observations suggest that GRK2 and GRK6 may contribute to the regulation of uterine contractility at term. Further work is necessary to determine whether GRKs and receptor desensitisation play a role in disorders of uterine contractility.

    Funded by: Wellcome Trust

    The Journal of endocrinology 1999;162;3;401-8

  • Role of G protein-coupled receptor kinases on the agonist-induced phosphorylation and internalization of the follitropin receptor.

    Lazari MF, Liu X, Nakamura K, Benovic JL and Ascoli M

    Department of Pharmacology, The University of Iowa College of Medicine, Iowa City 52242-1109, USA.

    The experiments presented herein were designed to identify members of the G protein-coupled receptor kinase (GRK) family that participate in the agonist-induced phosphorylation and internalization of the rat FSH receptor (rFSHR). Western blots of human kidney 293 cells (the cell line used in transfection experiments) and MSC-1 cells (a cell line derived from Sertoli cells that displays many of the differentiated functions of their normal counterparts) reveal the presence of GRK2 and GRK6 in both cell lines as well as GRK4 in MSC-1 cells. Cotransfection of 293 cells with the rFSHR and GRK2, GRK4alpha, or GRK6 resulted in an increase in the agonist-induced phosphorylation of the rFSHR. Cotransfections of the rFSHR with GRKs or arrestin-3 enhanced the agonist-induced internalization of the rFHSR, and combinations of GRKs and arrestin-3 were more effective than the individual components. To characterize the involvement of endogenous GRKs on phosphorylation and internalization, we inhibited endogenous GRK2 by overexpression of a kinase-deficient mutant of GRK2 or G alpha t, a scavenger of G betagamma. We also inhibited endogenous GRK6 by overexpression of a kinase-deficient mutant of GKR6. All three constructs were effective inhibitors of phosphorylation, but only the kinase-deficient mutant of GRK2 and G alpha t inhibited internalization. The inhibition of internalization induced by these two constructs was less pronounced than that induced by a dominant-negative mutant of the nonvisual arrrestins, however. The finding that inhibitors of GRK2 and GRK6 impair phosphorylation, but only the inhibitors of GRK2 impair internalization, suggests that different GRKs have differential effects on receptor internalization.

    Funded by: NICHD NIH HHS: HD-28962; NIGMS NIH HHS: GM-44944, GM-47417; ...

    Molecular endocrinology (Baltimore, Md.) 1999;13;6;866-78

  • Differential effects of CC chemokines on CC chemokine receptor 5 (CCR5) phosphorylation and identification of phosphorylation sites on the CCR5 carboxyl terminus.

    Oppermann M, Mack M, Proudfoot AE and Olbrich H

    Department of Immunology, University of Göttingen, 37075 Göttingen, Germany. mopperm@gwdg.de

    The binding of CC chemokines to CC chemokine receptor 5 (CCR5) triggers cellular responses that, generally, are only transient in nature. To explore the potential role of G protein-coupled receptor kinases (GRKs) in the regulation of CCR5, we performed phosphorylation experiments in a rat basophilic leukemia cell line stably expressing CCR5. The ability of various CCR5 ligands to stimulate calcium mobilization in these cells correlated with their ability to induce receptor phosphorylation, desensitization, internalization, and GRK association with the receptor. Aminooxypentane-RANTES, a potent inhibitor of human immunodeficiency virus infection, has been proposed to act through enhanced CCR5 internalization and inhibition of receptor recycling. Aminooxypentane-RANTES profoundly induced CCR5 phosphorylation, but had no effect on CCR1. In permeabilized rat basophilic leukemia CCR5 cells, monoclonal antibodies with specificity for GRK2/3 inhibited RANTES-induced receptor phosphorylation. Consistent with a role for these kinases in CCR5 regulation, 1-2 x 10(5) copies of GRK2 or GRK3 were found to be expressed in peripheral blood leukocytes. Phosphoamino acid analysis revealed that RANTES-induced CCR5 phosphorylation selectively occurs on serine residues. Our findings with receptor mutants indicate that serine residues at positions 336, 337, 342, and 349 represent GRK phosphorylation sites on CCR5. This study demonstrates that chemokines differ in their ability to induce CCR5 phosphorylation and desensitization and provides a molecular mechanism for the agonist-induced attenuation of CCR5 signaling.

    The Journal of biological chemistry 1999;274;13;8875-85

  • Regulation of G protein-coupled receptor kinases by caveolin.

    Carman CV, Lisanti MP and Benovic JL

    Departments of Biochemistry and Molecular Pharmacology and Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.

    G protein-coupled receptor kinases (GRKs) have been principally characterized by their ability to phosphorylate and desensitize G protein-coupled receptors. However, recent studies suggest that GRKs may have more diverse protein/protein interactions in cells. Based on the identification of a consensus caveolin binding motif within the pleckstrin homology domain of GRK2, we tested the direct binding of purified full-length GRK2 to various glutathione S-transferase-caveolin-1 fusion proteins, and we discovered a specific interaction of GRK2 with the caveolin scaffolding domain. Interestingly, analysis of GRK1 and GRK5, which lack a pleckstrin homology domain, revealed in vitro binding properties similar to those of GRK2. Maltose-binding protein caveolin and glutathione S-transferase-GRK fusion proteins were used to map overlapping regions in the N termini of both GRK2 and GRK5 that appear to mediate conserved GRK/caveolin interactions. In vivo association of GRK2 and caveolin was suggested by co-fractionation of GRK2 with caveolin in A431 and NIH-3T3 cells and was further supported by co-immunoprecipitation of GRK2 and caveolin in COS-1 cells. Functional significance for the GRK/caveolin interaction was demonstrated by the potent inhibition of GRK-mediated phosphorylation of both receptor and peptide substrates by caveolin-1 and -3 scaffolding domain peptides. These data reveal a novel mode for the regulation of GRKs that is likely to play an important role in their cellular function.

    Funded by: NCI NIH HHS: 5-T32-CA09662; NIGMS NIH HHS: GM44944, GM50443

    The Journal of biological chemistry 1999;274;13;8858-64

  • Sequestration of dopamine D2 receptors depends on coexpression of G-protein-coupled receptor kinases 2 or 5.

    Ito K, Haga T, Lameh J and Sadée W

    Department of Neurochemistry, Faculty of Medicine, University of Tokyo, Japan.

    We examined the agonist-dependent sequestration/internalization of dopamine D2 receptor (the long form D2L and short form D2S), which were transiently expressed in COS-7 and HEK 293 cells with or without G-protein-coupled receptor kinases (GRK2 or GRK5). Sequestration was assessed quantitatively by loss of [3H] sulpiride-binding activity from the cell surface and by transfer of [3H] spiperone-binding activity from the membrane fraction to the light vesicle fraction in sucrose-density gradients. In COS-7 cells expressing D2 receptors alone, virtually no sequestration was observed with or without dopamine (< 4%). When GRK2 was coexpressed, 50% of D2S receptors and 36% of D2L receptors were sequestered by treatment with 10(-4) M dopamine for 2 h, whereas no sequestration was observed in cells expressing the dominant negative form of GRK2 (DN-GRK2). When GRK5 was coexpressed, 36% of D2S receptors were sequestered following the same treatment. The agonist-dependent and GRK2-dependent sequestration of D2S receptors was reduced markedly in the presence of hypertonic medium containing 0.45 M sucrose, suggesting that the sequestration follows the clathrin pathway. Internalization of D2S receptors was also assessed by immunofluorescence confocal microscopy. Translocation of D2 receptors from the cell membrane to intracellular vesicles was observed following the treatment with dopamine from HEK 293 cells only when GRK2 was coexpressed. D2S receptors expressed in HEK 293 cells were shown to be phosphorylated by GRK2 in an agonist-dependent manner. These results indicate that the sequestration of D2 receptors occurs only through a GRK-mediated pathway.

    Funded by: NIDA NIH HHS: DA 04166; NIGMS NIH HHS: GM 43102; NIMH NIH HHS: MH 00996

    European journal of biochemistry 1999;260;1;112-9

  • beta2-Adrenergic receptor regulation by GIT1, a G protein-coupled receptor kinase-associated ADP ribosylation factor GTPase-activating protein.

    Premont RT, Claing A, Vitale N, Freeman JL, Pitcher JA, Patton WA, Moss J, Vaughan M and Lefkowitz RJ

    Departments of Medicine (Cardiology) and Biochemistry, Howard Hughes Medical Institute, Box 3821, Duke University Medical Center, Durham, NC 27710, USA.

    G protein-coupled receptor activation leads to the membrane recruitment and activation of G protein-coupled receptor kinases, which phosphorylate receptors and lead to their inactivation. We have identified a novel G protein-coupled receptor kinase-interacting protein, GIT1, that is a GTPase-activating protein (GAP) for the ADP ribosylation factor (ARF) family of small GTP-binding proteins. Overexpression of GIT1 leads to reduced beta2-adrenergic receptor signaling and increased receptor phosphorylation, which result from reduced receptor internalization and resensitization. These cellular effects of GIT1 require its intact ARF GAP activity and do not reflect regulation of GRK kinase activity. These results suggest an essential role for ARF proteins in regulating beta2-adrenergic receptor endocytosis. Moreover, they provide a mechanism for integration of receptor activation and endocytosis through regulation of ARF protein activation by GRK-mediated recruitment of the GIT1 ARF GAP to the plasma membrane.

    Funded by: NHLBI NIH HHS: HL16037, R01 HL016037

    Proceedings of the National Academy of Sciences of the United States of America 1998;95;24;14082-7

  • Expression of a beta-adrenergic receptor kinase 1 inhibitor prevents the development of myocardial failure in gene-targeted mice.

    Rockman HA, Chien KR, Choi DJ, Iaccarino G, Hunter JJ, Ross J, Lefkowitz RJ and Koch WJ

    Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. hrockman@med.unc.edu

    Heart failure is accompanied by severely impaired beta-adrenergic receptor (betaAR) function, which includes loss of betaAR density and functional uncoupling of remaining receptors. An important mechanism for the rapid desensitization of betaAR function is agonist-stimulated receptor phosphorylation by the betaAR kinase (betaARK1), an enzyme known to be elevated in failing human heart tissue. To investigate whether alterations in betaAR function contribute to the development of myocardial failure, transgenic mice with cardiac-restricted overexpression of either a peptide inhibitor of betaARK1 or the beta2AR were mated into a genetic model of murine heart failure (MLP-/-). In vivo cardiac function was assessed by echocardiography and cardiac catheterization. Both MLP-/- and MLP-/-/beta2AR mice had enlarged left ventricular (LV) chambers with significantly reduced fractional shortening and mean velocity of circumferential fiber shortening. In contrast, MLP-/-/betaARKct mice had normal LV chamber size and function. Basal LV contractility in the MLP-/-/betaARKct mice, as measured by LV dP/dtmax, was increased significantly compared with the MLP-/- mice but less than controls. Importantly, heightened betaAR desensitization in the MLP-/- mice, measured in vivo (responsiveness to isoproterenol) and in vitro (isoproterenol-stimulated membrane adenylyl cyclase activity), was completely reversed with overexpression of the betaARK1 inhibitor. We report here the striking finding that overexpression of this inhibitor prevents the development of cardiomyopathy in this murine model of heart failure. These findings implicate abnormal betaAR-G protein coupling in the pathogenesis of the failing heart and point the way toward development of agents to inhibit betaARK1 as a novel mode of therapy.

    Funded by: NHLBI NIH HHS: HL16037, HL46345, HL56687, P01 HL046345, P50 HL053773, R01 HL016037, R01 HL056687

    Proceedings of the National Academy of Sciences of the United States of America 1998;95;12;7000-5

  • Monocyte chemoattractant protein-1-induced CCR2B receptor desensitization mediated by the G protein-coupled receptor kinase 2.

    Aragay AM, Mellado M, Frade JM, Martin AM, Jimenez-Sainz MC, Martinez-A C and Mayor F

    Departamento de Biologia Molecular, Centro de Biologia Molecular "Severo Ochoa,", Universidad Autonoma de Madrid, Campus Cantoblanco, E-28049 Madrid, Spain.

    Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine cytokine family, whose physiological function is mediated by binding to the CCR2 and CCR4 receptors, which are members of the G protein-coupled receptor family. MCP-1 plays a critical role in both activation and migration of leukocytes. Rapid chemokine receptor desensitization is very likely essential for accurate chemotaxis. In this report, we show that MCP-1 binding to the CCR2 receptor in Mono Mac 1 cells promotes the rapid desensitization of MCP-1-induced calcium flux responses. This desensitization correlates with the Ser/Thr phosphorylation of the receptor and with the transient translocation of the G protein-coupled receptor kinase 2 (GRK2, also called beta-adrenergic kinase 1 or betaARK1) to the membrane. We also demonstrate that GRK2 and the uncoupling protein beta-arrestin associate with the receptor, forming a macromolecular complex shortly after MCP-1 binding. Calcium flux responses to MCP-1 in HEK293 cells expressing the CCR2B receptor were also markedly reduced upon cotransfection with GRK2 or the homologous kinase GRK3. Nevertheless, expression of the GRK2 dominant-negative mutant betaARK-K220R did not affect the initial calcium response, but favored receptor response to a subsequent challenge by agonists. The modulation of the CCR2B receptor by GRK2 suggests an important role for this kinase in the regulation of monocyte and lymphocyte response to chemokines.

    Proceedings of the National Academy of Sciences of the United States of America 1998;95;6;2985-90

  • The solution structure and dynamics of the pleckstrin homology domain of G protein-coupled receptor kinase 2 (beta-adrenergic receptor kinase 1). A binding partner of Gbetagamma subunits.

    Fushman D, Najmabadi-Haske T, Cahill S, Zheng J, LeVine H and Cowburn D

    Laboratory of Physical Biochemistry, The Rockefeller University, New York, New York 10021-6399, USA.

    The solution structure of an extended pleckstrin homology (PH) domain from the beta-adrenergic receptor kinase is obtained by high resolution NMR. The structure establishes that the beta-adrenergic receptor kinase extended PH domain has the same fold and topology as other PH domains, and there are several unique features, most notably an extended C-terminal alpha-helix that behaves as a molten helix, and a surface charge polarity that is extensively modified by positive residues in the extended alpha-helix and the C terminus. These observations complement biochemical evidence that the C-terminal portion of this PH domain participates in protein-protein interactions with Gbetagamma subunits. This suggests that the C-terminal segment of the PH domain may function to mediate protein-protein interactions with the targets of PH domains.

    Funded by: NCRR NIH HHS: RR-00862; NIGMS NIH HHS: GM-47021

    The Journal of biological chemistry 1998;273;5;2835-43

  • Phosphorylation and desensitization of human endothelin A and B receptors. Evidence for G protein-coupled receptor kinase specificity.

    Freedman NJ, Ament AS, Oppermann M, Stoffel RH, Exum ST and Lefkowitz RJ

    Howard Hughes Medical Institute, Department of Medicine, Duke University Medical Center, Box 3821, Durham, North Carolina 27710, USA.

    Although endothelin-1 can elicit prolonged physiologic responses, accumulating evidence suggests that rapid desensitization affects the primary G protein-coupled receptors mediating these responses, the endothelin A and B receptors (ETA-R and ETB-R). The mechanisms by which this desensitization proceeds remain obscure, however. Because some intracellular domain sequences of the ETA-R and ETB-R differ substantially, we tested the possibility that these receptor subtypes might be differentially regulated by G protein-coupled receptor kinases (GRKs). Homologous, or receptor-specific, desensitization occurred within 4 min both in the ETA-R-expressing A10 cells and in 293 cells transfected with either the human ETA-R or ETB-R. In 293 cells, this desensitization corresponded temporally with agonist-induced phosphorylation of each receptor, assessed by receptor immunoprecipitation from 32Pi-labeled cells. Agonist-induced receptor phosphorylation was not substantially affected by PKC inhibition but was reduced 40% (p < 0.03) by GRK inhibition, effected by a dominant negative GRK2 mutant. Inhibition of agonist-induced phosphorylation abrogated agonist-induced ETA-R desensitization. Overexpression of GRK2, -5, or -6 in 293 cells augmented agonist-induced ET-R phosphorylation approximately 2-fold (p < 0.02), but each kinase reduced receptor-promoted phosphoinositide hydrolysis differently. While GRK5 inhibited ET-R signaling by only approximately 25%, GRK2 inhibited ET-R signaling by 80% (p < 0.01). Congruent with its superior efficacy in suppressing ET-R signaling, GRK2, but not GRK5, co-immunoprecipitated with the ET-Rs in an agonist-dependent manner. We conclude that both the ETA-R and ETB-R can be regulated indistinguishably by GRK-initiated desensitization. We propose that because of its affinity for ET-Rs demonstrated by co-immunoprecipitation, GRK2 is the most likely of the GRKs to initiate ET-R desensitization.

    Funded by: NHLBI NIH HHS: HL03008, HL16037

    The Journal of biological chemistry 1997;272;28;17734-43

  • GRK2 and beta-arrestin 1 as negative regulators of thyrotropin receptor-stimulated response.

    Iacovelli L, Franchetti R, Masini M and De Blasi A

    Consorzio Mario Negri Sud, Istituto di Ricerche Farmacologiche Mario Negri, Santa Maria Imbaro, Italy.

    Arrestins are regulatory proteins for a number of G-coupled receptors. The binding of arrestin to receptor phosphorylated by G protein-coupled receptor kinase (GRK) quenches the activation of the G protein, thus resulting in receptor homologous desensitization. We have previously shown that the levels of beta-arrestin1 are regulated by intracellular cAMP and proposed that this may represent one homeostatic mechanism with which to regulate some cellular responses. To test this hypothesis, we focused on the TSH receptor using a rat thyroid cell line, FRTL5. We found that beta-arrestin1 is the only detectable isoform of arrestin expressed in FRTL5 and that its expression is regulated by TSH. To investigate the possible role of GRK2/beta-arrestin1 machinery in the mechanism of TSH receptor homologous desensitization, we used a cotransfection approach. The TSH-induced cAMP accumulation in COS7 cells transfected with TSH receptor was reduced by 35-45% when cotransfected with GRK2 and/or beta-arrestin1, indicating that the TSH receptor can be regulated by a GRK/arrestin mechanism. This raised the hypothesis that TSH increases the levels of beta-arrestin1, which in turn could regulate the TSH stimulation. To test this point a FRTL5-derived cell line overexpressing beta-arrestin1 was generated. In these cells the TSH-stimulated cAMP accumulation and, more importantly, the mitogenic activity were substantially blunted. Our results show that TSH receptor-stimulated cAMP accumulation and cell proliferation can be controlled by a GRK2/beta-arrestin1 mechanism.

    Molecular endocrinology (Baltimore, Md.) 1996;10;9;1138-46

  • Phosphorylation of the type 1A angiotensin II receptor by G protein-coupled receptor kinases and protein kinase C.

    Oppermann M, Freedman NJ, Alexander RW and Lefkowitz RJ

    Howard Hughes Medical Institute, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.

    The type 1A angiotensin II receptor (AT1A-R), which mediates cardiovascular effects of angiotensin II, has been shown to undergo rapid agonist-induced desensitization. We investigated the potential role of second messenger-activated kinases and G protein-coupled receptor kinases (GRKs) in the regulation of this receptor. In 293 cells transfected with the AT1A-R, a 3-min challenge with angiotensin II engendered a 46% decrease in subsequent angiotensin II-stimulated phosphoinositide hydrolysis in intact cells. This agonist-induced desensitization correlated temporally and dose-dependently with the phosphorylation of the receptor to a stoichiometry of 1 mol of phosphate/mol of receptor, as assessed by immunoprecipitation of receptors from cells metabolically labeled with 32Pi. Agonist-induced receptor phosphorylation was reduced by 40-50% by either overexpression of a dominant negative K220R mutant GRK2 or treatment of the cells with the protein kinase C (PKC) inhibitor staurosporine, in a virtually additive fashion. Cellular overexpression of GRK2K220R not only inhibited agonist-induced AT1A-R phosphorylation, but also prevented receptor desensitization, as assessed by angiotensin II-stimulated GTPase activity in membranes prepared from agonist-treated and control cells. In contrast, PKC inhibition by staurosporine did not affect homologous desensitization of the AT1A-R. Overexpression of GRKs 2, 3, or 5 significantly augmented the agonist-induced AT1A-R phosphorylation 1.5- to 1.7-fold (p < 0.001). These findings suggest a role for receptor phosphorylation by one or several GRKs in the rapid agonist-induced desensitization of the AT1A-R.

    Funded by: NHLBI NIH HHS: HL03008, HL16037

    The Journal of biological chemistry 1996;271;22;13266-72

  • Four consecutive serines in the third intracellular loop are the sites for beta-adrenergic receptor kinase-mediated phosphorylation and desensitization of the alpha 2A-adrenergic receptor.

    Eason MG, Moreira SP and Liggett SB

    Department of Pulmonary Medicine, University of Cincinnati College of Medicine, Ohio 45267-0564.

    During short term agonist exposure, the alpha 2A-adrenergic receptor (alpha 2AAR) undergoes rapid functional desensitization caused by phosphorylation of the receptor by the beta-adrenergic receptor kinase (beta ARK). This signal quenching is similar in nature to that found with a number of G-protein coupled receptors in which agonist-promoted desensitization is due to beta ARK phosphorylation; like these other receptors, the precise molecular determinants of the receptor required for beta ARK phosphorylation are not known. To delineate such a motif in the human alpha 2AAR (alpha 2C10), we constructed six mutated receptors consisting of deletions or substitutions of Ser-296-299 in the EESSSS sequence of the third intracellular loop of the receptor. These were expressed in Chinese hamster ovary and COS-7 cells, and agonist-promoted desensitization and receptor phosphorylation were assessed. Deletion of the EESSSS sequence and substitution of alanine for all four serines resulted in a total loss of phosphorylation and desensitization. Mutant receptors that retained two of the original serines (AASS and SSAA) underwent agonist-promoted phosphorylation of 55 +/- 7% and 57 +/- 8% of the phosphorylation found for wild type alpha 2C10. Additional substitution mutants (SSSA and SAAA) underwent 77 +/- 1% and 27 +/- 4% of wild type phosphorylation, respectively. Thus, substitution of alanine for each additional serine decreased overall phosphorylation as compared with wild type alpha 2C10 by approximately 25%, which is consistent with all 4 serines being phosphorylated. Mutated receptors that only partially phosphorylated (as compared with wild type) failed to undergo agonist-promoted desensitization. Thus, beta ARK-mediated phosphorylation of alpha 2C10 occurs at Ser-296-299 in the third intracellular loop, and this represents the critical step in rapid agonist-promoted desensitization. A number of other G-protein coupled receptors that undergo desensitization have a sequence motif similar to that which we have found for beta ARK-mediated phosphorylation of alpha 2C10, suggesting that these receptors may also be substrates for beta ARK.

    The Journal of biological chemistry 1995;270;9;4681-8

  • Phosphorylation of the N-formyl peptide receptor carboxyl terminus by the G protein-coupled receptor kinase, GRK2.

    Prossnitz ER, Kim CM, Benovic JL and Ye RD

    Department of Immunology, Scripps Research Institute, La Jolla, California 92037.

    Attenuation of receptor-mediated signal amplification in response to external stimuli, an essential step in the balance of cellular activation, may be mediated by receptor phosphorylation. We have recently shown that the carboxyl-terminal cytoplasmic domain of the N-formyl peptide receptor (FPR) interacts with G proteins and demonstrate here that this same region of the FPR is specifically phosphorylated by a neutrophil cytosolic kinase with properties similar to the G protein-coupled receptor kinase, GRK2. Both kinase activities show a lack of sensitivity toward protein kinase A, protein kinase C, and tyrosine kinase inhibitors but demonstrate almost identical sensitivity toward the kinase inhibitor heparin. Kinetic studies demonstrated that GRK2 has a Km for the carboxyl-terminal domain of the FPR of approximately 1.5 microM and that denaturation of the substrate results in an almost complete loss of phosphorylation. Comparative studies reveal that GRK3 has approximately 50% of the activity of GRK2 toward the FPR carboxyl terminus, whereas GRK5 and GRK6 have no detectable activity. Site-directed mutagenesis of numerous regions of the FPR carboxyl terminus demonstrated that, whereas Glu326/Asp327 and Asp333 are critical for phosphorylation, the carboxyl-terminal 10 amino acids are not required. Simultaneous substitution of Thr334, Thr336, Ser338, and Thr339 resulted in an approximately 50% reduction in phosphorylation, whereas simultaneous substitution of the upstream Ser328, Thr329, Thr331, and Ser332 or merely the Ser328 and Thr329 residues resulted in an approximately 80% reduction in phosphorylation. The introduction of negatively charged glutamate residues for Ser328 and Thr329 or Thr331 and Ser332 resulted in marked stimulation of phosphorylation. These results suggest a hierarchical mechanism in which phosphorylation of amino-terminal serine and threonine residues is required for the subsequent phosphorylation of carboxyl-terminal residues. These results provide the first direct evidence that an intracellular domain of a chemoattractant receptor is a high affinity substrate for GRK2 and further suggest a role for GRK2 or a closely related kinase in the attenuation of receptor-mediated activation of inflammatory cells.

    Funded by: NIAID NIH HHS: AI33503, AI36357; NIGMS NIH HHS: GM46572; ...

    The Journal of biological chemistry 1995;270;3;1130-7

  • Structure of the human gene encoding the beta-adrenergic receptor kinase.

    Penn RB and Benovic JL

    Department of Pharmacology, Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.

    The beta-adrenergic receptor kinase (beta ARK) specifically phosphorylates the agonist-occupied forms of the beta 2-adrenergic receptor and related G protein-coupled receptors. beta ARK is one of the best characterized members of a growing family of G protein-coupled receptor kinases. In this article we report the isolation and structural organization of the human beta ARK gene. The gene spans approximately 23 kilobases and is composed of 21 exons interrupted by 20 introns. Exon sizes range from 52 bases (exon 7) to over 1200 bases (exon 21), intron sizes from 68 bases (intron L) to 10.8 kilobases (intron A). The splice sites for donor and acceptor were in agreement with the canonical GT/AG rule. Functional regions of beta ARK are described with respect to their location within the exon-intron organization of the gene. Primer extension and RNase protection assays suggest a major transcription start site approximately 246 bases upstream of the start ATG. Sequence analysis of the 5'-flanking/promoter region reveals many features characteristic of mammalian housekeeping genes, i.e. the lack of a TATA box, an absent or nonstandard positioned CAAT box, high GC content, and the presence of Sp1-binding sites. The extraordinarily high GC content of the 5'-flanking region (> 80%) helps define this region as a CpG island that may be a principal regulator of beta ARK expression.

    The Journal of biological chemistry 1994;269;21;14924-30

  • High expression of beta-adrenergic receptor kinase in human peripheral blood leukocytes. Isoproterenol and platelet activating factor can induce kinase translocation.

    Chuang TT, Sallese M, Ambrosini G, Parruti G and De Blasi A

    Consorzio Mario Negri Sud, Istituto di Ricerche Farmacologiche Mario Negri, Santa Maria Imbaro, Italy.

    Receptor phosphorylation is a key step in the process of desensitization of the beta-adrenergic and other related receptors. A selective kinase (called beta-adrenergic receptor kinase, beta ARK) has been identified which phosphorylates the agonist-occupied form of the receptor. Recently the bovine beta ARK cDNA has been cloned and the highest levels of specific mRNA were found in highly innervated tissues. It was proposed that beta ARK may be primarily active on synaptic receptors. In the present study, the cDNA of human beta ARK was cloned and sequenced. The sequence was very similar to that of the bovine beta ARK (the overall amino acid homology was 98%). Very high levels of beta ARK mRNA and kinase activity were found in peripheral blood leukocytes and in several myeloid and lymphoid leukemia cell lines. Since agonist-induced beta ARK translocation is considered the first step involved in beta ARK-mediated homologous desensitization, we screened a number of G-protein-coupled receptor agonists for their ability to induce beta ARK translocation. In human mononuclear leukocytes, beta-AR agonist isoproterenol and platelet-activating factor were able to induce translocation of beta ARK from cytosol to membrane. After 20 min of exposure to isoproterenol (10 microM), the cytosolic beta ARK activity decreased to 61% of control, while membrane-associated beta ARK activity increased to 170%. 20-min exposure to platelet-activating factor (1 microM) reduced the cytosolic beta ARK activity to 42% of control with concomitant increase in membrane beta ARK activity to 214% of control. The high levels of beta ARK expression in human peripheral blood leukocytes together with the ability of isoproterenol and platelet-activating factor to induce beta ARK translocation, suggest a role for beta ARK in modulating some receptor-mediated immune functions.

    The Journal of biological chemistry 1992;267;10;6886-92

  • cDNA cloning and chromosomal localization of the human beta-adrenergic receptor kinase.

    Benovic JL, Stone WC, Huebner K, Croce C, Caron MG and Lefkowitz RJ

    Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA 19140.

    The beta-adrenergic receptor kinase (beta ARK) mediates agonist-dependent phosphorylation of the beta 2-adrenergic and related G protein-coupled receptors. A cDNA encoding bovine beta ARK has recently been isolated. In this work we have isolated a cDNA encoding human beta ARK from a retinal cDNA library. The cDNA encodes a protein of 689 amino acids with an overall 98.0% amino acid and 92.5% nucleotide identity with bovine beta ARK. Chromosomal location of the human beta ARK gene was determined by correlating the presence of the beta ARK locus with retention of a specific human chromosome in a rodent-human hybrid panel. This analysis revealed that the human beta ARK locus segregated with the long arm of chromosome 11, centromeric to 11q13.

    Funded by: NCI NIH HHS: CA21124, CA39860; NIGMS NIH HHS: GM44944

    FEBS letters 1991;283;1;122-6

Gene lists (5)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
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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