EC:2.7.11.16 - FACTA Search

Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.16 (GRK5)
1,127 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Adenosine 5'-diphosphate (ADP) plays a central role in regulating platelet function by the activation of the G protein-coupled receptors P2Y(1) and P2Y(12). Although it is well established that aggregation responses of platelets to ADP desensitize, the underlying mechanisms involved remain unclear. In this study we demonstrate that P2Y(1)- and P2Y(12)-mediated platelet responses desensitize rapidly. Furthermore, we have established that these receptors desensitize by different kinase-dependent mechanisms. G protein-coupled receptor kinase (GRK) 2 and GRK6 are both endogenously expressed in platelets. Transient overexpression of dominant-negative mutants of these kinases or reductions in endogenous GRK expression by the use of specific siRNAs in 1321N1 cells showed that P2Y(12), but not P2Y(1), desensitization is mediated by GRKs. In contrast, desensitization of P2Y(1), but not P2Y(12), is largely dependent on protein kinase C activity. This study is the first to show that both P2Y(1) and P2Y(12) desensitize in human platelets, and it reveals ways in which their sensitivity to ADP may be differentially and independently altered.
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PMID:P2Y1 and P2Y12 receptors for ADP desensitize by distinct kinase-dependent mechanisms. 1566 14

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.
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PMID:G protein-coupled receptor kinase 2-mediated phosphorylation of ezrin is required for G protein-coupled receptor-dependent reorganization of the actin cytoskeleton. 1584 35

G protein-coupled receptor kinases (GRKs) are regulatory enzymes involved in the modulation of seven-transmembrane-helix receptors. In order to develop specific inhibitors for these kinases, we synthesized and investigated peptide inhibitors derived from the sequence of the first intracellular loop of the beta2-adrenergic receptor. Introduction of changes in the sequence and truncation of N- and C-terminal amino acids increased the inhibitory potency by a factor of 40. These inhibitors not only inhibited the prototypical GRK2 but also GRK3 and GRK5. In contrast there was no inhibition of protein kinase C and protein kinase A even at the highest concentration tested. The peptide with the sequence AKFERLQTVTNYFITSE inhibited GRK2 with an IC50 of 0.6 microM, GRK3 with 2.6 microM and GRK5 with 1.6 microM. The peptide inhibitors were non-competitive for receptor and ATP. These findings demonstrate that specific peptides can inhibit GRKs in the submicromolar range and suggest that a further decrease in size is possible without losing the inhibitory potency.
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PMID:Peptide inhibitors of G protein-coupled receptor kinases. 1610 34

Angiotensin II (Ang II) activates a wide spectrum of signaling responses via the AT1 receptor (AT1R) that mediate its physiological control of blood pressure, thirst, and sodium balance and its diverse pathological actions in cardiovascular, renal, and other cell types. Ang II-induced AT1R activation via Gq/11 stimulates phospholipases A2, C, and D, and activates inositol trisphosphate/Ca2+ signaling, protein kinase C isoforms, and MAPKs, as well as several tyrosine kinases (Pyk2, Src, Tyk2, FAK), scaffold proteins (G protein-coupled receptor kinase-interacting protein 1, p130Cas, paxillin, vinculin), receptor tyrosine kinases, and the nuclear factor-kappaB pathway. The AT1R also signals via Gi/o and G11/12 and stimulates G protein-independent signaling pathways, such as beta-arrestin-mediated MAPK activation and the Jak/STAT. Alterations in homo- or heterodimerization of the AT1R may also contribute to its pathophysiological roles. Many of the deleterious actions of AT1R activation are initiated by locally generated, rather than circulating, Ang II and are concomitant with the harmful effects of aldosterone in the cardiovascular system. AT1R-mediated overproduction of reactive oxygen species has potent growth-promoting, proinflammatory, and profibrotic actions by exerting positive feedback effects that amplify its signaling in cardiovascular cells, leukocytes, and monocytes. In addition to its roles in cardiovascular and renal disease, agonist-induced activation of the AT1R also participates in the development of metabolic diseases and promotes tumor progression and metastasis through its growth-promoting and proangiogenic activities. The recognition of Ang II's pathogenic actions is leading to novel clinical applications of angiotensin-converting enzyme inhibitors and AT1R antagonists, in addition to their established therapeutic actions in essential hypertension.
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PMID:Pleiotropic AT1 receptor signaling pathways mediating physiological and pathogenic actions of angiotensin II. 1614 58

Oxytocin (OT) has long been used as an uterotonic during labor management in women, and yet responses to OT infusion remain variable and unpredictable among patients. The investigation of oxytocin receptor (OTR) regulation will benefit labor management, because the clinical practice of continuous iv infusion of OT is not optimal. As with other G protein-coupled receptors, it is likely that the OTR internalizes and/or desensitizes upon continuous agonist exposure. The mechanisms by which this might occur, however, are unclear. Here we explore OTR internalization and desensitization in human embryonic kidney cells by utilizing inhibitors of heterologous second messenger systems and recently available mutant cDNA constructs. We report rapid and extensive internalization and desensitization of the OTR upon agonist exposure. Internalization was unaffected by inhibitors of protein kinase C or Ca(2+) calmodulin-dependant kinase II but was significantly reduced after transfection with dominant-negative mutant cDNAs of G protein-coupled receptor kinase 2, beta-Arrestin2, Dynamin, and Eps15 (a component of clathrin-coated pits). Moreover, desensitization of the OTR, measured by a calcium mobilization assay, was also inhibited by the aforementioned cDNA constructs. Thus, our data demonstrate, for the first time, the importance of the classical clathrin-mediated pathway during agonist-induced OTR internalization and desensitization.
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PMID:Internalization and desensitization of the oxytocin receptor is inhibited by Dynamin and clathrin mutants in human embryonic kidney 293 cells. 1617 83

The G protein-coupled thyrotropin-releasing hormone (TRH) receptor is phosphorylated and binds to beta-arrestin after agonist exposure. To define the importance of receptor phosphorylation and beta-arrestin binding in desensitization, and to determine whether beta-arrestin binding and receptor endocytosis are required for receptor dephosphorylation, we expressed TRH receptors in fibroblasts from mice lacking beta-arrestin-1 and/or beta-arrestin-2. Apparent affinity for [(3)H]MeTRH was increased 8-fold in cells expressing beta-arrestins, including a beta-arrestin mutant that did not permit receptor internalization. TRH caused extensive receptor endocytosis in the presence of beta-arrestins, but receptors remained primarily on the plasma membrane without beta-arrestin. beta-Arrestins strongly inhibited inositol 1,4,5-trisphosphate production within 10 s. At 30 min, endogenous beta-arrestins reduced TRH-stimulated inositol phosphate production by 48% (beta-arrestin-1), 71% (beta-arrestin-2), and 84% (beta-arrestins-1 and -2). In contrast, receptor phosphorylation, detected by the mobility shift of deglycosylated receptor, was unaffected by beta-arrestins. Receptors were fully phosphorylated within 15 s of TRH addition. Receptor dephosphorylation was identical with or without beta-arrestins and almost complete 20 min after TRH withdrawal. Blocking endocytosis with hypertonic sucrose did not alter the rate of receptor phosphorylation or dephosphorylation. Expressing receptors in cells lacking Galpha(q) and Galpha(11) or inhibiting protein kinase C pharmacologically did not prevent receptor phosphorylation or dephosphorylation. Overexpression of dominant negative G protein-coupled receptor kinase-2 (GRK2), however, retarded receptor phosphorylation. Receptor activation caused translocation of endogenous GRK2 to the plasma membrane. The results show conclusively that receptor dephosphorylation can take place on the plasma membrane and that beta-arrestin binding is critical for desensitization and internalization.
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PMID:Beta-arrestin mediates desensitization and internalization but does not affect dephosphorylation of the thyrotropin-releasing hormone receptor. 1618 93

Dopamine, via activation of D1-like receptors, inhibits Na,K-ATPase and Na,H-exchanger in renal proximal tubules and promotes sodium excretion. This effect of dopamine is not seen in conditions associated with oxidative stress such as hypertension, diabetes, and aging due to uncoupling of D1-like receptors from G proteins. To identify the role of oxidative stress in uncoupling of the D1-like receptors, we utilized primary cultures from rat renal proximal tubules. Hydrogen peroxide (H2O2), an oxidant, treatment to the cell cultures increased the level of malondialdehyde, a marker of oxidative damage. Further, H2O2 decreased membranous D1-like receptor numbers and proteins, D1-like agonist (SKF 38393)-mediated [35S]GTPgammaS binding and SKF 38393-mediated inhibition of Na,K-ATPase. Moreover, H2O2 treatment to the cultures caused membranous translocation of G-protein-coupled receptor kinase 2 (GRK 2) and increased serine phosphorylation of D1A receptors accompanied by an increase in protein kinase C (PKC) activity. Interestingly, PKC inhibitors blocked the H2O2-mediated stimulation of GRK 2 and serine phosphorylation of D1A receptors. Further, GRK 2 antisense but not scrambled oligonucleotides attenuated the effect of H2O2 on membranous expression of GRK 2. Moreover, direct activation of PKC with phorbol ester (PMA) resulted in reduction of SKF 38393-mediated [35S]GTPgammaS binding. We conclude that H2O2 stimulates PKC leading to the activation of GRK 2, which causes serine phopshorylation of D1A receptors and receptor G-protein uncoupling in these cells, resulting in impairment in D1-like receptor function.
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PMID:Hydrogen peroxide causes uncoupling of dopamine D1-like receptors from G proteins via a mechanism involving protein kinase C and G-protein-coupled receptor kinase 2. 1633 75

A growing body of evidences suggests that receptor desensitization is implicated in the development of tolerance to opioids, which is generally regulated by protein kinases and receptor trafficking proteins. In the present study, we demonstrated that repeated s.c. treatment with etorphine, but not morphine, produced a significant increase in protein levels of G protein-coupled receptor kinase 2, dynamin II, beta-arrestin 2 and phosphorylated-conventional protein kinase C in membranes of the mouse spinal cord, suggesting that the etorphine-induced mu-opioid receptor desensitization may result from G protein-coupled receptor kinase 2/dynaminII/beta-arrestin2-dependent phosphorylation of mu-opioid receptors. Unlike etorphine, morphine failed to change the levels of these trafficking proteins. Furthermore, we found that the level of glial fibrillary acidic protein in the mouse spinal cord was clearly increased by chronic in vivo and in vitro treatment with morphine, whereas no such effect was noted by etorphine. In the behavioral study, intraperitoneal pretreatment with the glial-modulating agent propentofylline suppressed the development of tolerance to morphine-induced antinociception. In addition, intrathecal injection of astrocytes and astrocyte-conditioned medium mixture, which were obtained from cultured astrocytes of the newborn mouse spinal cord, aggravated the development of tolerance to morphine. In contrast, these agents failed to affect the development of tolerance induced by etorphine. These findings provide direct evidence for the distinct mechanisms between etorphine and morphine on the development of tolerance to spinal antinociception. These findings raise the possibility that the increased astroglia response produced by chronic morphine could be associated with the lack of mu-opioid receptor internalization.
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PMID:mu-Opioid receptor internalization-dependent and -independent mechanisms of the development of tolerance to mu-opioid receptor agonists: Comparison between etorphine and morphine. 1641 75

The ability of two opioid agonists, [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) and morphine, to induce mu-opioid receptor (MOR) phosphorylation, desensitization, and internalization was examined in human embryonic kidney (HEK) 293 cells expressing rat MOR1 as well G protein-coupled inwardly rectifying potassium channel (GIRK) channel subunits. Both DAMGO and morphine activated GIRK currents, but the maximum response to DAMGO was greater than that of morphine, indicating that morphine is a partial agonist. The responses to DAMGO and morphine desensitized rapidly in the presence of either drug. Expression of a dominant negative mutant G protein-coupled receptor kinase 2 (GRK2), GRK2-K220R, markedly attenuated the DAMGO-induced desensitization of MOR1, but it had no effect on morphine-induced MOR1 desensitization. In contrast, inhibition of protein kinase C (PKC) either by the PKC inhibitory peptide PKC (19-31) or staurosporine reduced MOR1 desensitization by morphine but not that induced by DAMGO. Morphine and DAMGO enhanced MOR1 phosphorylation over basal. The PKC inhibitor bisindolylmaleimide 1 (GF109203X) inhibited MOR1 phosphorylation under basal conditions and in the presence of morphine, but it did not inhibit DAMGO-induced phosphorylation. DAMGO induced arrestin-2 translocation to the plasma membrane and considerable MOR1 internalization, whereas morphine did not induce arrestin-2 translocation and induced very little MOR1 internalization. Thus, DAMGO and morphine each induce desensitization of MOR1 signaling in HEK293 cells but by different molecular mechanisms; DAMGO-induced desensitization is GRK2-dependent, whereas morphine-induced desensitization is in part PKC-dependent. MORs desensitized by DAMGO activation are then readily internalized by an arrestin-dependent mechanism, whereas those desensitized by morphine are not. These data suggest that opioid agonists induce different conformations of the MOR that are susceptible to different desensitizing and internalization processes.
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PMID:Agonist-selective mechanisms of mu-opioid receptor desensitization in human embryonic kidney 293 cells. 1668 5

Neuronal excitability is inhibited by somatostatin, which might play important roles in seizure and neuroprotection. The possibility of whether the effect of somatostatin on neurotransmission is susceptible to desensitization was investigated. We tested the effects of prolonged exposure to somatostatin on 0.1 mM extracellular Mg(2+) concentration ([Mg(2+)](o))-induced intracellular free Ca(2+) concentration ([Ca(2+)](i)) spikes in cultured rat hippocampal neurons using fura-2-based microfluorimetry. Reducing [Mg(2+)](o) to 0.1 mM elicited repetitive [Ca(2+)](i) spikes. These [Ca(2+)](i) spikes were inhibited by exposure to somatostatin-14. The inhibitory effects of somatostatin were blocked by pretreatment with pertussis toxin (PTX, 100 ng/ml) for 18-24 h. Prolonged exposure to somatostatin induced a desensitization of the somatostatin-induced inhibition of [Ca(2+)](i) spikes in a concentration-dependent manner. The somatostatin-induced desensitization was retarded by the nonspecific protein kinase C (PKC) inhibitor staurosporin (100 nM) or chronic treatment with phorbol dibutyrate (1 microM) for 24 h, but not by the protein kinase A inhibitor KT5720. The desensitization was significantly retarded by the novel PKCepsilon translocation inhibitor peptide (1 microM). In addition, suramin (3 microM), an inhibitor of G-protein-coupled receptor kinase 2 (GRK2), caused a reduction in the desensitization. After tetrodotoxin (TTX, 1 microM) completely blocked the low [Mg(2+)](o)-induced [Ca(2+)](i) spikes, glutamate-induced [Ca(2+)](i) transients were slightly inhibited by somatostatin and the inhibition was desensitized by prolonged exposure to somatostatin. These results indicate that the prolonged activation of somatostatin receptors induces the desensitization of somatostatin-induced inhibition on low [Mg(2+)](o)-induced [Ca(2+)](i) spikes through the activation of GRK2 and partly a novel PKCepsilon in cultured rat hippocampal neurons.
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PMID:Desensitization of somatostatin-induced inhibition of low extracellular magnesium concentration-induced calcium spikes in cultured rat hippocampal neurons. 1687 4

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