Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Persistent stimulation of the beta 1-adrenergic receptor (beta 1AR) engenders, within minutes, diminished responsiveness of the beta 1 AR/adenylyl cyclase signal transduction system. This desensitization remains incompletely defined mechanistically, however. We therefore tested the hypothesis that agonist-induced desensitization of the beta 1AR (like that of the related beta 2AR) involves phosphorylation of the receptor itself, by cAMP-dependent protein kinase (PKA) and the beta-adrenergic receptor kinase (beta ARK1) or other G protein-coupled receptor kinases (GRKs). Both Chinese hamster fibroblast and 293 cells demonstrate receptor-specific desensitization of the beta 1 AR within 3-5 min. Both cell types also express beta ARK1 and the associated inhibitory proteins beta-arrestin-1 and beta-arrestin-2, as assessed by immunoblotting. Agonist-induced beta 1AR desensitization in 293 cells correlates with a 2 +/- 0.3-fold increase in phosphorylation of the beta 1AR, determined by immunoprecipitation of the beta 1AR from cells metabolically labeled with 32P(i). This agonist-induced beta 1AR phosphorylation derives approximately equally from PKA and GRK activity, as judged by intact cell studies with kinase inhibitors or dominant negative beta ARK1 (K220R) mutant overexpression. Desensitization, likewise, is reduced by only approximately 50% when PKA is inhibited in the intact cells. Overexpression of rhodopsin kinase, beta ARK1, beta ARK2, or GRK5 significantly increases agonist-induced beta 1AR phosphorylation and concomitantly decreases agonist-stimulated cellular cAMP production (p < 0.05). Furthermore, purified beta ARK1, beta ARK2, and GRK5 all demonstrate agonist-dependent phosphorylation of the beta 1AR. Consistent with a GRK mechanism, receptor-specific desensitization of the beta 1AR was enhanced by overexpression of beta-arrestin-1 and -2 in transfected 293 cells. We conclude that rapid agonist-induced desensitization of the beta 1AR involves phosphorylation of the receptor by both PKA and at least beta ARK1 in intact cells. Like the beta 2AR, the beta 1AR appears to bind either beta-arrestin-1 or beta-arrestin-2 and to react with rhodopsin kinase, beta ARK1, beta ARK2, and GRK5.
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PMID:Phosphorylation and desensitization of the human beta 1-adrenergic receptor. Involvement of G protein-coupled receptor kinases and cAMP-dependent protein kinase. 762 2

The glucagon receptor is a member of the G protein-coupled receptor superfamily. Since several G protein-coupled receptors undergo phosphorylation in response to agonist, we investigated the phosphorylation of the glucagon receptor following the addition of glucagon to a Chinese hamster ovary cell line expressing the human glucagon receptor (CHO/hGR). Glucagon induced a rapid, time and concentration-dependent phosphorylation of its receptor on serine residues. Neither forskolin nor phorbol ester increased receptor phosphorylation, suggesting that cAMP-dependent protein kinase and protein kinase C do not catalyze this phosphorylation event. Furthermore, two mutant cell lines expressing glucagon receptors with successively truncated receptor cytoplasmic tails were tested. A strong correlation between the number of potential phosphorylation sites, receptor phosphorylation and receptor internalization was observed, suggesting that phosphorylation of the glucagon receptor in CHO/hGR cells is functionally linked to its internalization.
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PMID:Glucagon induces a rapid and sustained phosphorylation of the human glucagon receptor in Chinese hamster ovary cells. 860 65

SPARC, a counteradhesive matricellular protein, inhibits endothelial cell adhesion and proliferation, but the pathways through which these activities are blocked are not known. In this study, we used inhibitors of major signaling proteins to identify mediators through which SPARC exerts its counteradhesive and antiproliferative functions. Pretreatments with the general protein tyrosine kinase (PTK) inhibitors, herbimycin A and genistein, protected against the inhibitory effect of SPARC on bovine aortic endothelial (BAE) cell spreading by more than 60%. Similar pretreatments with PTK inhibitors significantly blocked the diminishment of focal adhesions by SPARC in confluent BAE cell monolayers, as determined by the formation of actin stress-fibers and the distribution of vinculin in focal adhesion plaques. Inhibition of endothelial cell cycle progression by SPARC and a calcium-binding SPARC peptide, however, was not affected by PTK inhibitors. Inhibition of DNA synthesis by SPARC was not reversed by inhibitors of the activity of protein kinase C (PKC), or of cAMP-dependent protein kinase (PKA), but was sensitive to pertussis (and to a lesser extent, cholera) toxin. The counteradhesive effect of SPARC on endothelial cells is, therefore, mediated through a tyrosine phosphorylation-dependent pathway, whereas its antiproliferative function is dependent, in part, on signal transduction via a G protein-coupled receptor.
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PMID:SPARC inhibits endothelial cell adhesion but not proliferation through a tyrosine phosphorylation-dependent pathway. 971 51

Promiscuous coupling between G protein-coupled receptors and multiple species of heterotrimeric G proteins provides a potential mechanism for expanding the diversity of G protein-coupled receptor signaling. We have examined the mechanism and functional consequences of dual Gs/Gi protein coupling of the beta3-adrenergic receptor (beta3AR) in 3T3-F442A adipocytes. The beta3AR selective agonist disodium (R, R)-5-[2[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1, 3-benzodioxole-2,2-dicarboxylate (CL316,243) stimulated a dose-dependent increase in cAMP production in adipocyte plasma membrane preparations, and pretreatment of cells with pertussis toxin resulted in a further 2-fold increase in cAMP production by CL316,243. CL316,243 (5 microM) stimulated the incorporation of 8-azido-[32P]GTP into Galphas (1.57 +/- 0.12; n = 3) and Galphai (1. 68 +/- 0.13; n = 4) in adipocyte plasma membranes, directly demonstrating that beta3AR stimulation results in Gi-GTP exchange. The beta3AR-stimulated increase in 8-azido-[32P]GTP labeling of Galphai was equivalent to that obtained with the A1-adenosine receptor agonist N6-cyclopentyladenosine (1.56 +/- 0.07; n = 4), whereas inclusion of unlabeled GTP (100 microM) eliminated all binding. Stimulation of the beta3AR in 3T3-F442A adipocytes led to a 2-3-fold activation of mitogen-activated protein (MAP) kinase, as measured by extracellular signal-regulated kinase-1 and -2 (ERK1/2) phosphorylation. Pretreatment of cells with pertussis toxin (PTX) eliminated MAP kinase activation by beta3AR, demonstrating that this response required receptor coupling to Gi. Expression of the human beta3AR in HEK-293 cells reconstituted the PTX-sensitive stimulation of MAP kinase, demonstrating that this phenomenon is not exclusive to adipocytes or to the rodent beta3AR. ERK1/2 activation by the beta3AR was insensitive to the cAMP-dependent protein kinase inhibitor H-89 but was abolished by genistein and AG1478. These data indicate that constitutive beta3AR coupling to Gi proteins serves both to restrain Gs-mediated activation of adenylyl cyclase and to initiate additional signal transduction pathways, including the ERK1/2 MAP kinase cascade.
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PMID:The beta3-adrenergic receptor activates mitogen-activated protein kinase in adipocytes through a Gi-dependent mechanism. 1020 24

The endocytic pathway of the secretin receptor, a class II GPCR, is unknown. Some class I G protein-coupled receptors (GPCRs), such as the beta(2)-adrenergic receptor (beta(2)-AR), internalize in clathrin-coated vesicles and this process is mediated by G protein-coupled receptor kinases (GRKs), beta-arrestin, and dynamin. However, other class I GPCRs, for example, the angiotensin II type 1A receptor (AT(1A)R), exhibit different internalization properties than the beta(2)-AR. The secretin receptor, a class II GPCR, is a GRK substrate, suggesting that like the beta(2)-AR, it may internalize via a beta-arrestin and dynamin directed process. In this paper we characterize the internalization of a wild-type and carboxyl-terminal (COOH-terminal) truncated secretin receptor using flow cytometry and fluorescence imaging, and compare the properties of secretin receptor internalization to that of the beta(2)-AR. In HEK 293 cells, sequestration of both the wild-type and COOH-terminal truncated secretin receptors was unaffected by GRK phosphorylation, whereas inhibition of cAMP-dependent protein kinase mediated phosphorylation markedly decreased sequestration. Addition of secretin to cells resulted in a rapid translocation of beta-arrestin to plasma membrane localized receptors; however, secretin receptor internalization was not reduced by expression of dominant negative beta-arrestin. Thus, like the AT(1A)R, secretin receptor internalization is not inhibited by reagents that interfere with clathrin-coated vesicle-mediated internalization and in accordance with these results, we show that secretin and AT(1A) receptors colocalize in endocytic vesicles. This study demonstrates that the ability of secretin receptor to undergo GRK phosphorylation and beta-arrestin binding is not sufficient to facilitate or mediate its internalization. These results suggest that other receptors may undergo endocytosis by mechanisms used by the secretin and AT(1A) receptors and that kinases other than GRKs may play a greater role in GPCR endocytosis than previously appreciated.
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PMID:Properties of secretin receptor internalization differ from those of the beta(2)-adrenergic receptor. 1053 54

The gastrointestinal hormone, glucose-dependent insulinotropic polypeptide (GIP), is one of the most important regulators of insulin secretion following ingestion of a meal. GIP stimulates insulin secretion from the pancreatic beta-cell via its G protein-coupled receptor activation of adenylyl cyclase and other signal transduction pathways, but there is little known regarding subsequent protein kinase pathways that are activated. A screening technique was used to determine the relative abundance of 75 protein kinases in CHO-K1 cells expressing the GIP receptor and in two pancreatic beta-cell lines (betaTC-3 and INS-1 (832/13) cells). This information was used to identify kinases that are potentially regulated following GIP stimulation, with a focus on GIP regulation of the ERK1/2 MAPK pathway. In CHO-K1 cells, GIP induced phosphorylation of Raf-1 (Ser-259), Mek1/2 (Ser-217/Ser-221), ERK1/2 (Thr-202 and Tyr-204), and p90 RSK (Ser-380) in a concentration-dependent manner. Activation of ERK1/2 was maximal at 4 min and was cAMP-dependent protein kinase-dependent and protein kinase C-independent. Studies using a beta-cell line (INS-1 clone 832/13) corroborated these findings, and it was also demonstrated that the ERK1/2 module could be activated by GIP in the absence of glucose. Finally, we have shown that GIP regulation of the ERK1/2 module is via Rap1 but does not involve Gbetagamma subunits nor Src tyrosine kinase, and we propose that cAMP-based regulation occurs via B-Raf in both CHO-K1 and beta-cells. These results establish the importance of GIP in the cellular regulation of the ERK1/2 module and identify a role for cAMP in coupling its G protein-coupled receptors to ERK1/2 activity in pancreatic beta-cells.
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PMID:Glucose-dependent insulinotropic polypeptide activates the Raf-Mek1/2-ERK1/2 module via a cyclic AMP/cAMP-dependent protein kinase/Rap1-mediated pathway. 1213 4

It has been suggested that the cannabinoid receptor type 1 (CB1), a G protein-coupled receptor, is internalized after agonist binding and activation of the second messenger pathways. It is proposed that phosphorylation enhances the down-regulation of the CB1 receptor, thus contributing to tolerance. Alterations in phosphorylation of proteins in the signal transduction cascade after CB1receptor activation could also alter tolerance to cannabinoids. We addressed our hypothesis by evaluating the role of several kinases in antinociceptive tolerance to Delta(9)-tetrahydrocannabinol (THC). We evaluated cAMP-dependent protein kinase (PKA) using KT5720, a PKA inhibitor; protein kinase C (PKC) using bisindolylmaleimide I, HCl (bis), a PKC inhibitor; cGMP-dependent protein kinase (PKG) using KT5823, a PKG inhibitor; beta-adrenergic receptor kinase (beta-ARK) using low molecular weight heparin (LMWH), a beta-ARK inhibitor; and phosphatidylinositol-3 kinase (PI3-K) using 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), a PI3-K inhibitor and PP1, a Src family tyrosine kinase inhibitor. The cAMP analog used was dibutyryl-cAMP and the cGMP analog used was dibutyryl-cGMP. Our data indicate that selective kinases may be involved in cannabinoid tolerance. Mice and rats were rendered tolerant to Delta(9)-THC. The PKG inhibitor KT5823, the beta-ARK inhibitor LMWH, the PI3-K inhibitor LY294002, and inhibition of PKC by bis had no effect on tolerance. At a higher dose, bis attenuated the antinociceptive effect of delta(9)-THC in nontolerant mice. PP1, the Src family tyrosine kinase inhibitor, and KT5720, the PKA inhibitor, reversed THC-induced tolerance. In addition, inhibition of PKA reversed a decrease in dynorphin release shown to accompany THC tolerance in rats. These data support a role for PKA and Src tyrosine kinase in phosphorylation events in delta(9)-THC-tolerant mice.
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PMID:The role of several kinases in mice tolerant to delta 9-tetrahydrocannabinol. 1260 57

Specificity of transduction events is controlled at the molecular level by scaffold, anchoring, and adaptor proteins, which position signaling enzymes at proper subcellular localization. This allows their efficient catalytic activation and accurate substrate selection. A-kinase anchoring proteins (AKAPs) are group of functionally related proteins that compartmentalize the cAMP-dependent protein kinase (PKA) and other signaling enyzmes at precise subcellular sites in close proximity to their physiological substrate(s) and favor specific phosphorylation events. Recent evidence suggests that AKAP transduction complexes play a key role in regulating G protein-coupled receptor (GPCR) signaling. Regulation can occur at multiple levels because AKAPs have been shown both to directly modulate GPCR function and to act as downstream effectors of GPCR signaling. In this minireview, we focus on the molecular mechanisms through which AKAP-signaling complexes modulate GPCR transduction cascades.
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PMID:Regulation of g protein-coupled receptor signaling by a-kinase anchoring proteins. 1711 2

Acylation-stimulating protein (ASP), a lipogenic hormone, stimulates triglyceride (TG) synthesis and glucose transport upon activation of C5L2, a G protein-coupled receptor. ASP-deficient mice have reduced adipose tissue mass due to increased energy expenditure despite increased food intake. The objective of this study was to evaluate the blocking of ASP-C5L2 interaction via neutralizing antibodies (anti-ASP and anti-C5L2-L1 against C5L2 extracellular loop 1). In vitro, anti-ASP and anti-C5L2-L1 blocked ASP binding to C5L2 and efficiently inhibited ASP stimulation of TG synthesis and glucose transport. In vivo, neither anti-ASP nor anti-C5L2-L1 altered body weight, adipose tissue mass, food intake, or hormone levels (insulin, leptin, and adiponectin), but they did induce a significant delay in TG clearance [P < 0.0001, 2-way repeated-measures (RM) ANOVA] and NEFA clearance (P < 0.0001, 2-way RM ANOVA) after a fat load. After treatment with either anti-ASP or anti-C5L2-L1 antibody there was no change in adipose tissue AMPK activity, but neutralizing antibodies decreased perirenal TG mass (-38.4% anti-ASP, -18.8% anti-C5L2, P < 0.01-0.001) and perirenal LPL activity (-75.6% anti-ASP, -72.5% anti-C5L2, P < 0.05). In liver, anti-C5L2-L1 decreased TG mass (-42.8%, P < 0.05), whereas anti-ASP increased AMPK activity (+34.6%, P < 0.001). In the muscle, anti-C5L2-L1 significantly increased TG mass (+128.0%, P < 0.05), LPL activity (+226.1%, P < 0.001), and AMPK activity (+71.1%, P < 0.01). In addition, anti-ASP increased LPL activity (+164.4, P < 0.05) and AMPK activity (+53.9%, P < 0.05) in muscle. ASP/C5L2-neutralizing antibodies effectively block ASP-C5L2 interaction, altering lipid distribution and energy utilization.
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PMID:Acylation-stimulating protein/C5L2-neutralizing antibodies alter triglyceride metabolism in vitro and in vivo. 1771 93

Gonadal steroid production is stimulated by gonadotropin binding to G protein-coupled receptors (GPCRs). Although GPCR-mediated increases in intracellular cAMP are known regulators of steroidogenesis, the roles of other signaling pathways in mediating steroid production are not well characterized. Recent studies suggest that luteinizing hormone (LH) receptor activation leads to trans-activation of epidermal growth factor (EGF) receptors in the testes and ovary. This pathway is critical for LH-induced steroid production in ovarian follicles, probably through matrix metalloproteinase (MMP)-mediated release of EGF receptor (EGFR) binding ectodomains. Here we examined LH and EGF receptor cross-talk in testicular steroidogenesis using mouse MLTC-1 Leydig cells. We demonstrated that, similar to the ovary, trans-activation of the EGF receptor was critical for gonadotropin-induced steroid production in Leydig cells. LH-induced increases in cAMP and cAMP-dependent protein kinase (PKA) activity mediated trans-activation of the EGF receptor and subsequent mitogen-activated protein kinase (MAPK) activation, ultimately leading to StAR phosphorylation and mitochondrial translocation. Steroidogenesis in Leydig cells was unaffected by MMP inhibitors, suggesting that cAMP and PKA trans-activated EGF receptors in an intracellular fashion. Interestingly, although cAMP was always needed for steroidogenesis, the EGFR/MAPK pathway was activated and necessary only for early (30-60 min), but not late (120 min or more), LH-induced steroidogenesis in vitro. In contrast, 36-h EGF receptor inhibition in vivo significantly reduced serum testosterone levels in male mice, demonstrating the physiologic importance of this cross-talk. These results suggest that GPCR-EGF receptor cross-talk is a conserved regulator of gonadotropin-induced steroidogenesis in the gonads, although the mechanisms of EGF receptor trans-activation may vary.
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PMID:Cross-talk between G protein-coupled and epidermal growth factor receptors regulates gonadotropin-mediated steroidogenesis in Leydig cells. 1870 61


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