Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Angiotensin II (Ang II) induces hypertrophy of cardiac myocytes and hyperplasia of cardiac fibroblasts. To determine the molecular mechanism by which Ang II displayed different effects on cardiac myocytes and fibroblasts, we examined signal transduction pathways leading to activation of extracellular signal-regulated kinases (ERKs). Ang II-induced ERK activation was abolished by pretreatment with pertussis toxin and by overexpression of the Gbetagamma subunit-binding domain of the beta-adrenergic receptor kinase 1 in cardiac fibroblasts but not in cardiac myocytes. Inhibition of protein kinase C strongly inhibited activation of ERKs by Ang II in cardiac myocytes, whereas inhibitors of tyrosine kinases but not of protein kinase C abolished Ang II-induced ERK activation in cardiac fibroblasts. Overexpression of C-terminal Src kinase (Csk), which inactivates Src family tyrosine kinases, suppressed the activation of transfected ERK in cardiac fibroblasts. Ang II rapidly induced phosphorylation of Shc and association of Shc with Grb2. Cotransfection of the dominant-negative mutant of Ras or Raf-1 kinase abolished Ang II-induced ERK activation in cardiac fibroblasts. Overexpression of Csk or the dominant-negative mutant of Ras had no effects on Ang II-induced ERK activation in cardiac myocytes. These findings suggest that Ang II-evoked signal transduction pathways differ among cell types. In cardiac fibroblasts, Ang II activates ERKs through a pathway including the Gbetagamma subunit of Gi protein, tyrosine kinases including Src family tyrosine kinases, Shc, Grb2, Ras, and Raf-1 kinase, whereas Gq and protein kinase C are important in cardiac myocytes.
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PMID:Cell type-specific angiotensin II-evoked signal transduction pathways: critical roles of Gbetagamma subunit, Src family, and Ras in cardiac fibroblasts. 948 62

Tentative identification of the G protein-coupled receptor kinase 2 and 5 (GRK2 and GRK5) sites of phosphorylation of the beta2-adrenergic receptor (betaAR) was recently reported based on in vitro phosphorylation of recombinant receptor (Fredericks, Z. L., Pitcher, J. A., and Lefkowitz, R. J. (1996) J. Biol. Chem. 271, 13796-13803). Phosphorylated residues identified for GRK2 were threonine 384 and serines 396, 401, and 407. GRK5 phosphorylated these four residues as well as threonine 393 and serine 411. To determine if mutation of these sites altered desensitization, we have constructed betaARs in which the threonines and serines of the putative GRK2 and GRK5 sites were substituted with alanines. These constructs were further modified to eliminate the cAMP-dependent protein kinase (PKA) consensus sites. Mutants betaARs were transfected into HEK 293 cells, and standard kinetic parameters were measured following 10 microM epinephrine treatment of cells. The mutant and wild type (WT) receptors were all desensitized 89-94% after 5 min of 10 microM epinephrine stimulation and 96-98% after a 30-min pretreatment. There were no significant changes observed for any of the mutant betaARs relative to the WT in the extent of 10 microM epinephrine-induced internalization (77-82% after 30 min). Epinephrine treatment for 1 min induced a rapid increase in the phosphorylation of the GRK5 and PKA- mutant betaARs as well as the WT. We conclude that sites other than the GRK2 and GRK5 sites identified by in vitro phosphorylation are involved in mediating the major effects of the in vivo GRK-dependent desensitization of the betaAR.
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PMID:Desensitization of beta2-adrenergic receptors with mutations of the proposed G protein-coupled receptor kinase phosphorylation sites. 951 68

beta2-Adrenergic receptors (beta2ARs) are important regulators of airway smooth muscle tone, and beta-sympathomimetic drugs are the most widely used agents in asthma therapy and are universally recognized as the treatment of choice for acute asthma attacks. Despite the clinical importance of beta-agonists and a good understanding of their mechanism of action in airway smooth muscle relaxation, surprisingly little is known about the manner in which the beta2AR signaling pathway is regulated in human airway smooth muscle (HASM). In this communication, we characterize mechanisms underlying rapid desensitization of the HASM beta2AR-adenylyl cyclase (AC) pathway. Acute homologous desensitization of beta2AR-mediated cyclic adenosine monophosphate (cAMP) production was characterized by an approximately 60% loss of maximal responsiveness to isoproterenol (ISO) when cells were pretreated for 30 min with 1 microM ISO. Acute heterologous beta2AR desensitization was characterized by an approximately 20% and 30% loss of maximal responsiveness to ISO challenge when cells were pretreated with forskolin and prostaglandin E2 (PGE2), respectively. Each form of desensitization was also characterized by an increase in the EC50 for ISO. beta2AR sequestration was associated with but not required for homologous desensitization. However, sequestration was required for rapid resensitization. Minimal alterations in inherent AC activity were observed with both modes of desensitization, suggesting that the beta2AR is the principal locus of regulation. Protein kinase inhibition by staurosporine largely reversed heterologous beta2AR desensitization and had a small but significant effect on homologous desensitization. In contrast, bisindolylmaleimide IX, a specific PKC-inhibitor, had no effect on heterologous or homologous beta2AR desensitization, suggesting that staurosporine effects were mediated by PKA inhibition. Overexpression of the G protein-coupled receptor kinase GRK2 in HASM cultures enhanced homologous desensitization. These data suggest that HASM beta2ARs are highly susceptible to rapid desensitization by multiple agents, and identify both GRKs and PKA as important mediators of acute beta2AR desensitization.
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PMID:Mechanisms of acute desensitization of the beta2AR-adenylyl cyclase pathway in human airway smooth muscle. 969 8

In NG108-15 cells inhibition of both N-type calcium channel current and adenylyl cyclase by somatostatin (SRIF) was not sustained but rapidly desensitized in the continued presence of the drug. The degree and rate of desensitization were concentration-dependent, and the desensitization was homologous with respect to the delta-opioid receptor. We have been unable to obtain evidence for the involvement of G protein-coupled receptor kinases (GRKs) in this desensitization. SRIF-induced desensitization of N-type calcium channel currents was not reduced in cells stably overexpressing a dominant negative mutant of GRK2 or following intracellular dialysis with GRK2- and GRK3-blocking peptides or with heparin. Inhibitors of protein kinase A, protein kinase C, and protein kinase G were also without effect. In contrast, both the rate and degree of SRIF-induced desensitization were reduced by pretreatment with phenylarsine oxide or concanavalin A, both inhibitors of receptor endocytosis. Furthermore, SRIF-induced desensitization was enhanced by monensin, which prevents receptor recycling back to the plasma membrane. Similarly, SRIF-induced desensitization of adenylyl cyclase inhibition was not reduced in cells stably overexpressing dominant negative mutant GRK2 but was reduced in cells pretreated with the receptor endocytosis inhibitor hyperosmotic sucrose or concanavalin A. These data are consistent with the view that SRIF-induced desensitization in NG108-15 cells results from receptor internalization.
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PMID:Somatostatin receptor desensitization in NG108-15 cells. A consequence of receptor sequestration. 983 85

This investigation was undertaken to study the mechanisms of calcitonin gene-related peptide (CGRP)-mediated desensitization using recombinant porcine CGRP receptors stably expressed in human embryonic kidney (HEK-293) cells. Pretreatment of these cells with human alphaCGRP resulted in an approximately 60% decrease in CGRP-stimulated adenylyl cyclase activity and an approximately 10-fold rightward shift in the dose-response curve of CGRP. This effect was rapid (t(1/2) approximately 5 min) and was accompanied by a significant decrease in [125I]CGRP binding to membrane preparations from CGRP-pretreated cells. In contrast, CGRP pretreatment had no effect on isoproterenol- or forskolin-stimulated adenylyl cyclase activity in these cells. The potential involvement of protein kinase A or protein kinase C in CGRP-mediated desensitization was studied using selective inhibitors or activators of these kinases. Pretreatment of the cells with forskolin (adenylyl cyclase activator) or phorbol dibutyrate (protein kinase C activator) had no effect on CGRP-mediated adenylyl cyclase activity and did not influence CGRP-mediated desensitization. However, pretreatment of the cells with 2-(8-[(dimethylamino)methyl]-6,7,8, 9-tetrahydropyrido[1,2-a]indol-3-yl]-3-(1-methylindol-3-yl)m aleimide hydrochloride (Ro 32-0432) (a potent inhibitor of protein kinase C) resulted in significant attenuation of CGRP-mediated desensitization with an IC(50) approximately 3 microM. To establish whether this effect might be due to inhibition of other protein kinases by Ro 32-0432, its effect was tested against several G protein-coupled receptor kinases (GRKs). Ro 32-0432 was found to inhibit GRK2, GRK5, and GRK6 with IC(50) values of 29, 3.6, and 16 microM, respectively, suggesting that its effect on CGRP-mediated desensitization might be a result of GRK inhibition. To further test this hypothesis, as well as the potential GRK specificity, the cells were treated with antisense oligonucleotides to GRK2, GRK5, and GRK6. While GRK2 and GRK5 antisense nucleotides had no effect on CGRP-mediated desensitization, the GRK6 antisense nucleotide treatment significantly reversed CGRP-mediated desensitization. These results suggest the involvement of GRK6 in CGRP-mediated desensitization in HEK-293 cells.
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PMID:Involvement of G protein-coupled receptor kinase-6 in desensitization of CGRP receptors. 1096 37

The ability of the cytoplasmic, full-length C-terminus of the beta 2-adrenergic receptor (BAC1) expressed in Escherichia coli to act as a functional domain and substrate for protein phosphorylation was tested. BAC1 was expressed at high-levels, purified, and examined in solution as a substrate for protein phosphorylation. The mobility of BAC1 on SDS-PAGE mimics that of the native receptor itself, displaying decreased mobility upon chemical reduction of disulfide bonds. Importantly, the C-terminal, cytoplasmic domain of the receptor expressed in E. coli was determined to be a substrate for phosphorylation by several candidate protein kinases known to regulate G-protein-linked receptors. Mapping was performed by proteolytic degradation and matrix-assisted laser desorption ionization, time-of-flight mass spectrometry. Purified BAC1 is phosphorylated readily by protein kinase A, the phosphorylation occurring within the predicted motif RRSSSK. The kinetic properties of the phosphorylation by protein kinase A displayed cooperative character. The activated insulin receptor tyrosine kinase, which phosphorylates the beta-adrenergic receptor in vivo, phosphorylates BAC1. The Y364 residue of BAC1 was predominantly phosphorylated by the insulin receptor kinase. GRK2 catalyzed modest phosphorylation of BAC1. Phosphorylation of the human analog of BAC1 in which Cys341 and Cys378 were mutated to minimize disulfide bonding constraints, displayed robust phosphorylation following thermal activation, suggesting under standard conditions that the population of BAC1 molecules capable of assuming the "activated" conformer required by GRKs is low. BAC1 was not a substrate for protein kinase C, suggesting that the canonical site in the second cytoplasmic loop of the intact receptor is preferred. The functional nature of BAC1 was tested additionally by expression of BAC1 protein in human epidermoid carcinoma A431 cells. BAC1 was found to act as a dominant-negative, blocking agonist-induced desensitization of the beta-adrenergic receptor when expressed in mammalian cells. Thus, the C-terminal, cytoplasmic tail of this G-protein-linked receptor expressed in E. coli acts as a functional domain, displaying fidelity with regard to protein kinase action in vivo and acting as a dominant-negative with respect to agonist-induced desensitization.
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PMID:The full-length, cytoplasmic C-terminus of the beta 2-adrenergic receptor expressed in E. coli acts as a substrate for phosphorylation by protein kinase A, insulin receptor tyrosine kinase, GRK2, but not protein kinase C and suppresses desensitization when expressed in vivo. 1108 85

Pituitary adenylyl cyclase-activating polypeptide (PACAP) receptor type 1 (PAC(1)) signaling and desensitization were investigated in human retinoblastoma Y-79 cells. Concentration-dependent stimulation of cAMP accumulation was observed in Y-79 cells incubated for 30 min with PACAP38, PACAP27, or VIP (10(-12) to 10(-6) M). The following EC(50) values were calculated: PACAP38, 24+/-3 pM; PACAP27, 99+/-8 pM; and VIP, 29+/-3 nM. Homologous desensitization of PAC(1) receptors in Y-79 cells pretreated with 10 nM PACAP38 or PACAP27 for 60 min was characterized by a 30-50% reduction in PACAP-stimulated cAMP accumulation (p<0.0001) and a two- to fivefold rightward shift in EC(50) values (p<0.0001). PAC(1) receptor desensitization was not accompanied by a reduction in PAC(1) mRNA expression. We concluded that the desensitizing effect of PACAP38 was homologous because neither corticotropin-releasing factor- nor (-)-isoproterenol-stimulated cAMP accumulation was altered by PACAP38 preincubation. Pretreating Y-79 cells with the protein kinase A (PKA) inhibitor H89 failed to inhibit homologous PAC(1) receptor desensitization. Similarly, pretreating Y-79 cells with the protein kinase C (PKC) inhibitors staurosporine or bisindolylmaleimide failed to alter homologous PAC(1) receptor desensitization. Although activation of PKA by dibutyryl cAMP or forskolin did not desensitize PAC(1) receptors, direct activation of PKC by PMA heterologously desensitized PAC(1) receptors, reducing cAMP accumulation 34.2+/-2.2% (p<0.001). Using RT-PCR, mRNA levels for G-protein-coupled receptor kinase 3 (GRK3), but not GRK2, were found to increase 2.2- to 4.8-fold in Y-79 cells exposed to PACAP38 for 10 min to 24 h (p<0.001). PAC(1) receptor desensitization decreased 72.5+/-4.3% (p<0.001) in Y-79 cells transfected with a GRK3 antisense cDNA construct that also reduced GRK3 protein expression 48.5+/-7.9% (p<0.0005). These experiments demonstrate that GRK3 plays an important role in the homologous desensitization of retinoblastoma PAC(1) receptors, whereas PKC, but not PKA, contributes to the heterologous desensitization of retinoblastoma PAC(1) receptors.
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PMID:G-protein-coupled receptor kinase 3- and protein kinase C-mediated desensitization of the PACAP receptor type 1 in human Y-79 retinoblastoma cells. 1116 32

Potential G protein-coupled receptor kinase (GRK) and protein kinase A (PKA) mediation of homologous desensitization of corticotropin-releasing factor type 1 (CRF1) receptors was investigated in human retinoblastoma Y-79 cells. Inhibition of PKA activity by PKI(5-22) or H-89 failed to attenuate homologous desensitization of CRF1 receptors, and direct activation of PKA by forskolin or dibutyryl cAMP failed to desensitize CRF-induced cAMP accumulation. However, treatment of permeabilized Y-79 cells with heparin, a nonselective GRK inhibitor, reduced homologous desensitization of CRF1 receptors by approximately 35%. Furthermore, Y-79 cell uptake of a GRK3 antisense oligonucleotide (ODN), but not of a random or mismatched ODN, reduced GRK3 mRNA expression by approximately 50% without altering GRK2 mRNA expression and inhibited homologous desensitization of CRF1 receptors by approximately 55%. Finally, Y-79 cells transfected with a GRK3 antisense cDNA construct exhibited an approximately 50% reduction in GRK3 protein expression and an ~65% reduction in homologous desensitization of CRF1 receptors. We conclude that GRK3 contributes importantly to the homologous desensitization of CRF1 receptors in Y-79 cells, a brain-derived cell line.
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PMID:GRK3 mediates desensitization of CRF1 receptors: a potential mechanism regulating stress adaptation. 1124 13

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.
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PMID:Regulation of membrane targeting of the G protein-coupled receptor kinase 2 by protein kinase A and its anchoring protein AKAP79. 1127 69

Receptor desensitization provides a potential mechanism for the regulation of adrenocortical adrenocorticotropin (ACTH) responsiveness. Using the mouse adrenocortical Y1 cell line we demonstrate that ACTH effectively desensitizes the cAMP response of its own receptor, the melanocortin 2 receptor (MC2R), in these cells with a maximal effect between 30 and 60 min. Neither forskolin nor isoproterenol (in Y1 cells stably transfected with the beta(2)-adrenergic receptor) desensitize this ACTH response. ACTH desensitizes its receptor at concentrations at which only a fraction of receptors are occupied, implying that this mechanism acts on agonist-unoccupied receptors. Y1 cells express G protein-coupled receptor kinase (GRK) 2 and 5, but stable expression of a dominant negative GRK2 (K220W) only marginally reduces the desensitization by ACTH. The protein kinase A (PKA) inhibitor, H89, extinguishes almost the entire desensitization response over the initial 30-min period at all concentrations of ACTH. A mutant MC2R in which the single consensus PKA phosphorylation site has been mutated (S208A) when expressed in MC2R-negative Y6 cells is also unable to desensitize. These data imply a heterologous, PKA-dependent, mode of desensitization, which is restricted to agonist-occupied and -unoccupied MC2R, possibly as a consequence of receptor/effector complexes that functionally compartmentalize this receptor.
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PMID:Desensitization of the Y1 cell adrenocorticotropin receptor: evidence for a restricted heterologous mechanism implying a role for receptor-effector complexes. 1157 4


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