Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cholecystokinin (CCK) receptor on the rat pancreatic acinar cell is a G protein-coupled receptor that is phosphorylated in response to homologous and heterologous agonist stimulation. In this work we have studied the stoichiometry of receptor phosphorylation and have utilized one-dimensional phosphopeptide mapping after cyanogen bromide cleavage to demonstrate that the third intracellular loop is the predominant domain of phosphorylation of this receptor in response to these treatments. Of the average 5 mol of phosphate/mol of receptor, greater than 95% was on the third loop, with the remainder residing on the carboxyl-terminal tail. Serine residues were the site of greater than 95% of phosphorylation, with threonine representing the remainder, and no phosphotyrosine was detected. Further, we have utilized two-dimensional phosphopeptide mapping after subtilisin cleavage to identify differing sites of CCK receptor phosphorylation which are dependent on the agonist utilized to stimulate this cell. Both qualitative and quantitative differences in phosphorylation sites were observed after acinar cell stimulation with different protein kinase C agonists. Further, distinct phosphopeptides on the map were identified as representing substrate(s) of a staurosporine-insensitive kinase activity stimulated only by receptor occupation with native CCK and were felt to represent site(s) of action of a member of the G protein-coupled receptor kinase family. This represents a sensitive and powerful approach that is applicable to sparse receptors residing in their native cellular environment to assess possible differences in patterns of phosphorylation which may be important in agonist-specific receptor regulation.
 ...
PMID:Phosphopeptide mapping of cholecystokinin receptors on agonist-stimulated native pancreatic acinar cells. 753 8

With chronic opiate use, opioid receptor desensitization may be one of the important mechanisms underlying the development of opiate tolerance and addiction. Opioid receptors belong to the G protein-coupled receptor superfamily. In this study, the mouse delta-opioid receptor (delta OR) was used in a model system to investigate the role of opioid receptor phosphorylation in receptor desensitization. When expressed in 293 cells and exposed to agonist, the delta OR underwent receptor-specific desensitization within 10 min. This agonist-induced desensitization corresponded temporally to a 3-fold increase in receptor phosphorylation. Phorbol ester, but not forskolin, also stimulated phosphorylation of the delta OR in 293 cells. Although down-regulation of protein kinase C failed to affect agonist-induced receptor phosphorylation, it abolished phorbol ester-induced receptor phosphorylation. Agonist-induced delta OR phosphorylation must therefore involve kinases other than protein kinase C. Whereas overexpression of a dominant negative mutant (K220R) of beta-adrenergic receptor kinase-1 (beta ARK1) in 293 cells significantly reduced agonist-dependent phosphorylation of the delta OR, overexpression of beta ARK1 or G protein-coupled receptor kinase-5 significantly enhanced this phosphorylation. Concordantly, beta ARK1-K220R overexpression reduced agonist-dependent delta OR desensitization, whereas beta ARK1 overexpression enhanced this densensitization. We conclude that short term desensitization of the delta OR involves phosphorylation of the receptor by one or more G protein-coupled receptor kinases.
 ...
PMID:Agonist-dependent phosphorylation of the mouse delta-opioid receptor: involvement of G protein-coupled receptor kinases but not protein kinase C. 765 49

The objectives of the present study were to determine whether the G protein-coupled receptor for PTH and PTH-related protein (PTHrP) is subject to agonist-specific phosphorylation and to characterize the relevant kinase(s). The opossum kidney PTH/PTHrP receptor stably expressed in human embryonic kidney 293 cells was coupled to adenylyl cyclase, with half-maximal activation occurring in the presence of 0.1 nM bovine (b) PTH-(1-34). Immunoprecipitation of extracts of 32P-labeled cells using a monoclonal antibody to the PTH/PTHrP receptor revealed the presence of a major 32P-labeled protein of approximately 85 kilodaltons that was not evident in untransfected 293 cells. bPTH-(1-34) treatment produced a rapid dose-dependent increase in phosphorylation of the 85-kilodalton receptor, with a maximal effect that was 3.5 +/- 0.7-fold (n = 4) over basal. Half-maximal phosphorylation occurred with 10 nM bPTH-(1-34), similar to the hormone concentration required for 50% receptor occupancy. Activation of protein kinase A or protein kinase C with forskolin or phorbol 12-myristate 13-acetate also increased PTH/PTHrP receptor phosphorylation, but to a lesser degree than PTH. Neither of these kinases mediated the effect of PTH, as blockade of the protein kinase A pathway (with H-89) or the protein kinase C pathway (with the bisindolylmaleimide GF 109203X) did not inhibit bPTH-(1-34)-induced PTH/PTHrP receptor phosphorylation. These results suggest that agonist-stimulated PTH/PTHrP receptor phosphorylation may involve a nonsecond messenger-activated kinase, such as a member of the G protein-coupled receptor kinase family.
 ...
PMID:Agonist-stimulated phosphorylation of the G protein-coupled receptor for parathyroid hormone (PTH) and PTH-related protein. 766 44

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.
 ...
PMID:Phosphorylation of the N-formyl peptide receptor carboxyl terminus by the G protein-coupled receptor kinase, GRK2. 783 71

Human muscarinic acetylcholine receptor m1 subtypes (m1 receptors) were expressed in and purified from insect Sf9 cells and then subjected to phosphorylation by G protein-coupled receptor kinase 2 (GRK2) expressed in and purified from Sf9 cells and by protein kinase C purified from rat brain (a mixture of alpha, beta, and gamma types, PKC). The m1 receptor was phosphorylated by either GRK2 or PKC in an agonist-dependent or independent manner, respectively. G protein beta gamma subunits stimulated the phosphorylation by GRK2 but did not affect the phosphorylation by PKC. The number of incorporated phosphates was 4.6 and 2.8 mol/mol of receptor for phoshorylation by GRK2 and PKC, respectively. The number of incorporated phosphates was 7.5 mol/mol receptor for phosphorylation by GRK2 followed by PKC, but was 5.8 mol/mol of receptor for the phosphorylation by PKC followed by GRK2. Major sites phosphorylated by GRK2 and PKC were located in the third intracellular loop and the carboxyl-terminal tail, respectively. These results indicate that GRK2 and PKC phosphorylate different sites of m1 receptors and that the phosphorylation by PKC partially inhibits the phosphorylation by GRK2, probably by affecting activation of GRK2 by agonist-bound receptors.
 ...
PMID:Phosphorylation of human m1 muscarinic acetylcholine receptors by G protein-coupled receptor kinase 2 and protein kinase C. 857 54

Heterotrimeric G proteins (peripheral proteins) conduct signals from membrane receptors (integral proteins) to regulatory proteins localized to various cellular compartments. They are in excess over any G protein-coupled receptor type on the cell membrane, which is necessary for signal amplification. These facts account for the large number of G protein molecules bound to membrane lipids. Thus, the protein-lipid interactions are crucial for their cellular localization, and consequently for signal transduction. In this work, the binding of G protein subunits to model membranes (liposomes), formed with defined membrane lipids, has been studied. It is shown that although G protein alpha-subunits were able to bind to lipid bilayers, the presence of nonlamellar-prone phospholipids (phosphatidylethanolamines) enhanced their binding to model membranes. This mechanism also appears to be used by other (structurally and functionally unrelated) peripheral proteins, such as protein kinase C and the insect protein apolipophorin III, indicating that it could constitute a general mode of protein-lipid interactions, relevant in the activity and translocation of some peripheral (amphitropic) proteins from soluble to particulate compartments. Other factors, such as the presence of cholesterol or the vesicle surface charge, also modulated the binding of the G protein subunits to lipid bilayers. Conversely, the binding of G protein-coupled receptor kinase 2 and the G protein beta-subunit to liposomes was not increased by hexagonally prone lipids. Their distinct interactions with membrane lipids may, in part, explain the different cellular localizations of all of these proteins during the signaling process.
 ...
PMID:Role of lipid polymorphism in G protein-membrane interactions: nonlamellar-prone phospholipids and peripheral protein binding to membranes. 932 17

To investigate mechanisms underlying the agonist-induced desensitization of the type 1A angiotensin II receptor (AT1A-R), we have stably expressed in Chinese hamster ovary (CHO) cells the wild-type receptor and truncated mutants lacking varying lengths of the cytoplasmic tail. Assay of inositol 1,4,5-trisphosphate (IP3) formation in response to agonist demonstrated that the truncated mutants T318, T328, and T348 lacking the last 42, 32, or 12 amino acid residues, respectively, couple with Gq protein with an efficiency similar to that of full-length receptors, whereas coupling of Gq protein was abolished in the T310 truncated mutant devoid of the carboxyl-terminal 50 amino acids. Exposure of CHO/AT1A-R cells expressing the wild-type AT1A-R to angiotensin II resulted in rapid and dose-dependent homologous desensitization of receptor-mediated IP3 formation, which was independent of the receptor internalization. Mastoparan, an activator of G protein-coupled receptor kinase (GRK), induced desensitization of the AT1A-R. The agonist-induced desensitization of the receptor was largely prevented by heparin, a potent inhibitor of GRK, whereas it was only partially attenuated by a protein kinase C (PKC)-specific inhibitor. The homologous or heterologous desensitization of the receptor was greatly impaired in the truncated mutants T318 and T328, lacking the Ser/Thr-rich (13 or 12 Ser/Thr residues) cytoplasmic tail of the AT1A-R. Deletion of the last two Ser residues, including one PKC consensus site in the receptor tail, prevented only phorbol 12-myristate 13-acetate-induced desensitization by 30%. Moreover, we found an agonist-induced translocation of a heparin-sensitive kinase activity. The angiotensin II-stimulated heparin-sensitive kinase could phosphorylate a thioredoxin fusion protein containing the entire AT1A-R cytoplasmic tail (N295 to E359), which lacks consensus phosphorylation sites for GRK1, GRK2, and GRK3. The heparin-sensitive kinase may not be GRK2, GRK3, or GRK6 expressed in CHO/AT1A-R cells, since angiotensin II did not induce translocation of these receptor kinases. Potential Ser/Thr phosphorylation sites located between S328 and S347 in the cytoplasmic tail of AT1A-R seem to play a critical role in the heterologous and homologous desensitization of the receptor. A heparin-sensitive kinase other than GRK2, GRK3, or GRK6 may be involved in the agonist-induced homologous desensitization of the AT1A-R.
 ...
PMID:Role of cytoplasmic tail of the type 1A angiotensin II receptor in agonist- and phorbol ester-induced desensitization. 952 56

Phosphorylation of G protein-coupled receptors is considered an important step during their desensitization. In SK-N-BE cells, recently presented as a pertinent model for the studies of the human delta-opioid receptor, pretreatment with the opioid agonist etorphine increased time-dependently the rate of phosphorylation of a 51-kDa membrane protein. Immunological characterization of this protein with an antibody, raised against the amino-terminal region of the cloned human delta-opioid receptor, revealed that it corresponded to the delta-opioid receptor. During prolonged treatment with etorphine, phosphorylation increased as early as 15 min to reach a maximum within 1 h. Phosphorylation and desensitization of adenylyl cyclase inhibition paralleled closely and okadaic acid inhibited the resensitization, a result strongly suggesting that phosphorylation of the delta-opioid receptor plays a prominent role in its rapid desensitization. The increase in phosphorylation of the delta-opioid receptor, as well as its desensitization, was not affected by H7, an inhibitor of protein kinase A and protein kinase C, but was drastically reduced by heparin or Zn2+, known to act as G protein-coupled receptor kinase (GRK) inhibitors. These results are the first to show, on endogenously expressed human delta-opioid receptor, that a close link exists between receptor phosphorylation and agonist-promoted desensitization and that desensitization involves a GRK.
 ...
PMID:Desensitization of the delta-opioid receptor correlates with its phosphorylation in SK-N-BE cells: involvement of a G protein-coupled receptor kinase. 957

We have investigated the potential for protein kinase C (PKC) to phosphorylate and desensitize the alpha2A-adrenergic receptor (alpha2AAR). In whole-cell phosphorylation studies, recombinantly expressed human alpha2AAR displayed an increase in phosphorylation after short-term exposure to 100 nM phorbol 12-myristate-13-acetate (PMA) that was blocked by preincubation with a PKC inhibitor. This increase in receptor phosphorylation over basal amounted to 172 +/- 40% in COS-7 cells and 201 +/- 40% in Chinese hamster ovary cells. In permanently transfected Chinese hamster fibroblast cells, PKC activation by brief exposure of the cells to PMA resulted in a marked desensitization of alpha2AAR function, amounting to a 68 +/- 4% decrease in the maximal agonist (UK14304)-stimulated intracellular calcium release. Such desensitization was blocked by the PKC inhibitor bisindolylmaleimide I and was not evoked by an inactive phorbol ester. The desensitization of this agonist response was not caused by PKC-mediated augmentation of G protein-coupled receptor kinase activity, because PMA-promoted desensitization of a mutated alpha2AAR that lacked G protein-coupled receptor kinase phosphorylation sites was identical to that of wild-type alpha2AAR. To test whether PKC phosphorylation is a mechanism by which alpha2AAR can be regulated by other receptors, the alpha1bAR was co-expressed with the alpha2AAR in Chinese hamster ovary cells. Upon selective activation of alpha1bAR, the function of alpha2AAR underwent a 53 +/- 5% desensitization. Thus, cellular events that result in PKC activation promote phosphorylation of the alpha2AAR and lead to substantial desensitization of receptor function. This heterologous regulation also represents a mechanism by which rapid crosstalk between the alpha2AAR and other receptors can occur.
 ...
PMID:Phosphorylation and functional desensitization of the alpha2A-adrenergic receptor by protein kinase C. 965 88

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.
 ...
PMID:Mechanisms of acute desensitization of the beta2AR-adenylyl cyclase pathway in human airway smooth muscle. 969 8


1 2 3 4 5 6 7 8 Next >>