Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

G protein-coupled receptor kinases (GRKs) and beta-arrestin-2 play a crucial role in the regulation of neurotransmitter receptors in brain. In this study, GRK2, GRK6, beta-arrestin-2 and associated regulatory proteins (Gbeta proteins and protein phosphatase (PP)-2A) were quantitated in human brains (immunodensity with specific antibodies) to assess for postmortem changes (pattern of protein degradation) and to investigate the effect of aging on these regulatory proteins as well as their subcellular distribution (cytosol and membrane fractions). In brain (prefrontal cortex, total homogenate) of healthy subjects (n=14) the immunodensities of GRK2 (r=-0.76), GRK6 (r=-0.64), beta-arrestin-2 (r=-0.57), Gbeta proteins (r=-0.59) and neurofilament (NF)-L (r=-0.64), but not PP-2A, declined markedly with the length of postmortem delay (PMD, 3-81 h). With these linear decay models, the average decreases per 12 h of PMD (from 12 to 72 h) were 7-11% for the various proteins. The immunodensities of GRK2 (r=-0.71), GRK6 (r=-0.61), and beta-arrestin-2 (r=-0.54) in human brain (n=12) also declined with aging (16 to 87 years) and the average decreases per decade (from 20 to 80 years) were 3-5%. In contrast, the immunodensities of PP-2A, Gbeta and NF-L in brain did not correlate significantly with the age of the subject at death (16-87 years). The immunodensities of GRK2/6 and beta-arrestin-2 showed marked individual variations and were strongly reduced after several freeze/thaw cycles. In the prefrontal cortex the subcellular distribution (cytosol/membrane) of the two GRKs differed markedly (GRK2: 60%/40%; GRK6: 5%/95%), and that of beta-arrestin-2 was as expected for a soluble protein (60%/40%). In brains of healthy subjects, the immunodensities of cytosolic GRK2 and beta-arrestin-2 correlated, respectively, with those of membrane-associated GRK2 (r=0.67, P=0.049, n=9) and membrane-associated beta-arrestin-2 (r=0.77, P=0.01, n=9). The results of this study emphasize the importance of examining relevant variables (PMD, age) and potential artifacts (individual variation, freeze-thawing effect) when designing signal transduction studies in neuropsychiatric disorders using the postmortem human brain.
Brain Res Mol Brain Res 2002 May 30
PMID:G protein-coupled receptor kinases, beta-arrestin-2 and associated regulatory proteins in the human brain: postmortem changes, effect of age and subcellular distribution. 1200 30

G protein-coupled receptor kinases (GRKs) phosphorylate opioid receptors, which eventually results in receptor sequestration. With respect to kappa-opioid receptors, it is known that internalization occurs in a species-specific manner. That is, the agonist-occupied human kappa-receptors will sequester whereas murine receptors fail to do so. This investigation concentrates on the internalization of kappa-opioid receptors, employing laser scanning microscopy as a major technique to examine receptor internalization in living cells. For this reason, we fused green fluorescence protein to kappa-receptors, and DsRed-fluorescent protein to GRK2 and GRK3. All fusion proteins retained their biologic activities. Permanent cell lines (HEK 293, NG 108-15) were transfected to express either green fluorescent kappa-receptors or to coexpress the tagged receptor and a specific GRK-DsRed construct. The localization of fluorescent receptors and GRKs was monitored by confocal microscopy before and after opioid exposure of transfected cells. Activation of the murine kappa-receptors triggers rapid translocation of tagged GRKs toward the cell membrane, but receptor internalization was not observed. The agonist-occupied human kappa-receptor also causes translocation of GRK2- and GRK3-DsRed, which was followed by the formation of vesicles carrying the green fluorescent kappa-receptors. Moreover, the green fluorescent vesicles consistently harbour red fluorescent GRK2 and GRK3, respectively. The phenomenon of kappa-receptor internalization as well as cointernalization of GRKs is blocked by phosducin, indicating a critical role of G protein-betagamma subunits for kappa-receptor sequestration. Comparing the effect of over-expressed GRK2 and GRK3 on sequestration of kappa-receptors, we conclude that GRK3 more strongly induces kappa-receptor internalization than GRK2.
Mol Pharmacol 2002 Jun
PMID:Visualizing preference of G protein-coupled receptor kinase 3 for the process of kappa-opioid receptor sequestration. 1202 6

In order to understand the modification of beta-adrenoceptor linked signal transduction by changes in the intracellular Ca2+, we examined the status of beta-adrenoceptors (beta-ARs), G-proteins and adenylyl cyclase (AC) in Ca2+-deficiency and Ca2+-overload by perfusing the isolated rat heart with Ca2+-free medium for 5 min and Ca2+-containing medium for 5 min following Ca2+-free perfusion, respectively. Ca2+-depletion caused not only an increase in basal, isoproterenol-, Gpp(NH)p-, NaF- and forskolin-stimulated AC activities but also produced an increase in the beta1-AR affinity and density as well as up-regulation of G(s)-protein function and uncoupling of G(i)-protein to AC. Ca2+-repletion for 5 min following 5 min Ca2+-free perfusion reversed the increased AC activities to varying degrees. The beta1-AR affinity was further increased upon Ca2+-repletion whereas its density was decreased. Ca2+-repletion also decreased protein content for AC and beta-AR kinase but augmented the changes in G(s)- and G(i)-protein functions. Although low Na+ medium perfusion during Ca2+-depletion prevented the changes in G-proteins during both Ca2+-depletion and Ca2+-repletion periods, the increased beta1-AR affinity and density as well as changes in AC activities due to Ca2+-depletion were not affected while alterations due to Ca2+-repletion were fully prevented. These results suggest that changes in Ca2+-homeostasis may represent a mechanism for alterations in the beta-adrenergic signal transduction pathway in the heart under pathological conditions.
Mol Cell Biochem 2002 Mar
PMID:Alterations of cardiac beta-adrenoceptor mechanisms due to calcium depletion and repletion. 1203 Mar 81

The myocardial beta-adrenergic receptor (betaAR) system plays a key role in dysfunctional signaling and physiology of the failing heart. Recently we described a murine model of dilated cardiomyopathy (DCM) produced by cardiac-specific expression of a dominant negative form of the CREB transcription factor (CREB(A133) mice). CREB(A133) mice display abnormalities within the betaAR signaling system including loss of inotropic reserve. Rapid desensitization of betaARs is mediated by the betaAR kinase (betaARK1), which is upregulated during heart failure. Inhibition of betaARK1 activity in the heart via expression of a peptide inhibitor (betaARKct) has been shown to enhance myocardial function and to "rescue" several animal models of heart failure. To determine the role of betaAR dysfunction in the progression of DCM in the CREB(A133) mice, we interbred them with mice expressing the betaARKct. Concurrent expression of the betaARKct peptide and CREB(A133) in mouse hearts resulted in the normalization of elevated betaARK1 levels. This biochemical change resulted in partial restoration of isoproterenol-stimulated adenylate cyclase activity as well as improvement in fractional shortening in response to betaAR stimulation. Interestingly, the progression of DCM and premature mortality was not altered. Therefore, the pathogenesis of DCM in CREB(A133) mice does not appear to involve abnormal betaAR signaling as a key element in its pathological progression and accordingly, the restoration of betaAR signaling is not sufficient to prevent the development and progression of all forms of heart failure.
J Mol Cell Cardiol 2002 Jun
PMID:Inhibition of betaARK1 restores impaired biochemical beta-adrenergic receptor responsiveness but does not rescue CREB(A133) induced cardiomyopathy. 1205 54

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.
Mol Pharmacol 2002 Aug
PMID:Oxidative stress decreases G protein-coupled receptor kinase 2 in lymphocytes via a calpain-dependent mechanism. 1213 Jun 91

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.
Mol Pharmacol 2002 Dec
PMID:Reduction of G protein-coupled receptor kinase 2 expression in U-937 cells attenuates H2 histamine receptor desensitization and induces cell maturation. 1243 19

In the present study, we examined the roles of G(12), G(13), G(q), and G(i) in endothelin-1-induced hypertrophic responses. Endothelin-1 stimulation activated extracellular signal-regulated kinase (ERK) and c-Jun NH(2)-terminal kinase (JNK) in cultured rat neonatal myocytes. The activation of JNK, but not ERK, was inhibited by the expression of carboxyl terminal regions of G alpha(12) and G alpha(13). JNK activation was also inhibited by expression of the G alpha(12)/G alpha(13)-specific inhibitor regulator of G protein signaling (RGS) domain of p115RhoGEF and the G alpha(q)-specific inhibitor RGS domain of the G protein-coupled receptor kinase 2 (GRK2-RGS). JNK activation was not, however, inhibited by expression of the carboxyl terminal region of G protein-coupled receptor kinase 2 (GRK2-ct), which is a G beta gamma-sequestering polypeptide. Additionally, JNK activation but not ERK activation was inhibited by the expression of C3 exoenzyme that inactivates small GTPase Rho. These results suggest that JNK activation by G alpha(12), G alpha(13), and G alpha(q) is involved in Rho. On the other hand, ERK activation was inhibited by pertussis toxin treatment, the receptor-G(i) uncoupler, and GRK2-ct. Thus, ERK was activated by G alpha(i)- and G beta gamma-dependent pathways. These results clearly demonstrate that differential pathways activate JNK and ERK.
Mol Pharmacol 2003 Mar
PMID:Differential requirement of G alpha12, G alpha13, G alphaq, and G beta gamma for endothelin-1-induced c-Jun NH2-terminal kinase and extracellular signal-regulated kinase activation. 1260 54

The aurora kinases are a novel oncogenic family of mitotic serine/threonine kinases (S/T kinases) that are overexpressed in a number of solid tumors, including pancreas and colorectal cancer. A PSI-BLAST search [National Center for Biotechnology Information (NCBI)] with the sequence of the S/T kinase domain of human aurora1 kinase [also known as AUR1, ARK2, AIk2, AIM-1, and STK12] and human aurora2 kinase (also known as AUR2, ARK1, AIK, BTAK, and STK15) showed a high sequence similarity to the three-dimensional structures of bovine cAMP-dependent kinase [Brookhaven Protein Data Bank code 1CDK], murine cAMP-dependent kinase (1APM), and Caenorhabditis elegans twitchin kinase (1KOA). When the aurora1 or aurora2 sequence was input into the tertiary structure prediction programs THREADER and 3D-PSSM (three-dimensional position-sensitive scoring matrix), the top structural matches were 1CDK, 1APM, and 1KOA, confirming that these domains are structurally conserved. The structural models of aurora1 and aurora2 were built using 1CDK as the template structure. Molecular dynamics and docking simulations, targeting the ATP binding site of aurora2 with adenylyl imidodiphosphate (AMP-PNP), staurosporine, and six small molecular S/T kinase inhibitors, identified active-site residues that interact with these inhibitors differentially. The docked structures of the aurora2-AMP-PNP and aurora2-staurosporine complexes indicated that the adenine ring of AMP-PNP and the indolocarbazole moiety of staurosporine have similar positions and orientations and provided the basis for the docking of the other S/T kinase inhibitors. Inhibitors with isoquinoline and quinazoline moieties were recognized by aurora2 in which H-89 and 6,7-dimethoxyquinazoline compounds exhibited high binding energies compared with that of staurosporine. The calculated binding energies for the docked small-molecule inhibitors were qualitatively consistent with the IC(50) values generated using an in vitro kinase assay. The aurora2 structural model provides a rational basis for site-directed mutagenesis of the active site; design of novel H-89, staurosporine, and quinazoline analogues; and the screening of the available chemical database for the identification of other novel, small-molecular entities.
Mol Cancer Ther 2003 Mar
PMID:Targeting aurora2 kinase in oncogenesis: a structural bioinformatics approach to target validation and rational drug design. 1265 23

We have recently shown that the binding of arrestin-3 to the lutropin receptor (LHR) is dependent mostly on receptor activation rather than on phosphorylation. The experiments presented here were designed to test the involvement of these two events in the association of arrestin-3 with the closely related follitropin receptor (FSHR). Activation of the FSHR leads to the phosphorylation of residues in the first and third intracellular loops. Mutation of the phosphorylation sites in the third intracellular loop of the rat (r) FSHR partially reduces phosphorylation but has no effect on arrestin-3 association. Mutation of the phosphorylation sites in the first intracellular loop abolishes phosphorylation and arrestin-3 association. Dominant-negative mutants of G protein-coupled receptor kinase (GRKs) 2 and 6 inhibit rFSHR phosphorylation to the same extent but only the dominant-negative mutant of GRK2 inhibits arrestin-3 association. Two mutations of the rFSHR (D389N and Y530F) that impair activation and abolish phosphorylation also impair arrestin-3 binding. GRK2 restores the phosphorylation of both mutants but it restores arrestin-3 association only to the D389N mutant. We conclude that, in contrast to the data obtained with the LHR, the association of arrestin-3 with the FSHR is dependent on receptor phosphorylation. The phosphorylation of the third intracellular loop residues is not needed for arrestin-3 association, however.
Mol Cell Endocrinol 2003 Jun 30
PMID:The association of arrestin-3 with the follitropin receptor depends on receptor activation and phosphorylation. 1285 Feb 88

The G protein-coupled receptor (GPCR) kinase GRK2 phosphorylates G protein-coupled receptors in an agonist-dependent manner. GRK2 activity is modulated through interactions of diverse domains of the kinase with G protein betagamma subunits, several lipids, anchoring proteins, and activated receptors. We report that kinase activity toward either GPCR (rhodopsin) or a synthetic peptide substrate is enhanced in the presence of GST-GRK2 fusion proteins or peptides corresponding to either N- or C-terminal sequences of GRK2. This direct stimulatory action of intrinsic domains on GRK2 activity does not add to the effect of other regulators, such as Gbetagamma subunits, and strongly suggests the existence of some mode of autoregulation. The existence of regulatory intramolecular interactions in GRK2 is supported by the facts that a C-terminal peptide protects the N-terminal region from proteolytic cleavage and that two domains of GRK2 independently coexpressed in cells associate as assessed by immunoprecipitation. Molecular modeling suggests that intramolecular interactions among the N-terminal, C-terminal and kinase domains would keep GRK2 in a constrained conformation characteristic of an inactive, basal state. Our model proposes that disruption of such intramolecular contacts by intermolecular interactions with regulatory proteins (mimicked by exogenously added kinase fragments in vitro) would promote the conformational changes required to bring about GRK2 translocation and activation.
Mol Pharmacol 2003 Sep
PMID:Involvement of intramolecular interactions in the regulation of G protein-coupled receptor kinase 2. 1292 Jan 99


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