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)

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

GRK6, a 66-kDa serine/threonine protein kinase, is a recently identified member of the G protein-coupled receptor kinase (GRK) family. GRKs are involved in the phosphorylation of seven-transmembrane receptors, a process mediating desensitization of signal transduction. An important feature of these enzymes is their membrane-associated nature, which for some members is stimulus-dependent. The structural basis for this membrane association previously has been shown in different members of the GRK family to include isoprenylation, G protein beta gamma-binding domains, and basic regions to provide electrostatic interactions with phospholipids. We provide evidence that another mechanism includes fatty acid acylation. GRK6, but not other GRKs tested, incorporated tritium after incubation with [3H]palmitate in Sf9 and in COS-7 cells overexpressing the kinase. The incorporated radioactivity was released from the protein by neutral hydroxylamine, indicating the presence of a thioester bond, and was confirmed as palmitic acid by high performance liquid chromatography analysis. Site-directed mutagenesis defined the region of palmitate attachment as a cluster of 3 cysteines (Cys561, Cys562, and Cys565) in the carboxyl-terminal domain of the kinase, consistent with the location of the membrane targeting domains of GRKs 1, 2, 3, and 5. Palmitoylation of GRK6 appears essential for membrane association, since palmitoylated kinase was found only in the membrane fraction. This lipid modification provides a structural basis for potential regulation of the subcellular distribution of GRK6 through acylation/deacylation cycles.
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PMID:Palmitoylation of G protein-coupled receptor kinase, GRK6. Lipid modification diversity in the GRK family. 796 2

G protein-coupled receptor kinases (GRK), such as the beta-adrenergic receptor kinase (beta ARK) and rhodopsin kinase, specifically phosphorylate the activated form of G protein-coupled receptors. To identify additional members of the GRK family, we screened a human heart cDNA library by low stringency hybridization using the catalytic domains of two beta ARK isoforms. Here we report the isolation of a cDNA that encodes a 576-amino-acid protein kinase, termed GRK6, that has significant homology with GRK5 (70.1% amino acid identity), IT11 (68.5%), rhodopsin kinase (47.1%), and beta ARK (37.4%). RNA blot analysis of GRK6 with selected human tissues reveals two distinct mRNAs of 3 and 2.4 kilobases with a distribution very similar to that of beta ARK (i.e. brain, skeletal muscle > pancreas > heart, lung, kidney, placenta > liver). GRK6, overexpressed in Sf9 insect cells using the baculovirus system, was able to phosphorylate both the beta 2-adrenergic receptor and rhodopsin in a stimulus-dependent fashion, although it was significantly less active then beta ARK on these substrates. These data extend the family of GRKs and suggest that GRK6 may have a substrate specificity quite distinct from beta ARK and rhodopsin kinase.
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PMID:Molecular cloning and expression of GRK6. A new member of the G protein-coupled receptor kinase family. 836 96

A novel human G protein-coupled receptor kinase was recently identified by positional cloning in the search for the Huntington's disease locus (Ambrose, C., James, M., Barnes, G., Lin, C., Bates, G., Altherr, M., Duyao, M., Groot, N., Church, D., Wasmuth, J. J., Lehrach, H., Housman, D., Buckler, A., Gusella, J. F., and MacDonald, M. E. (1993) Hum. Mol. Genet. 1, 697-703). Comparison of the deduced amino acid sequence of GRK4 with those of the closely related GRK5 and GRK6 suggested the apparent loss of 32 codons in the amino-terminal domain and 46 codons in the carboxyl-terminal domain of GRK4. These two regions undergo alternative splicing in the GRK4 mRNA, resulting from the presence or absence of exons filling one or both of these apparent gaps. Each inserted sequence maintains the open reading frame, and the deduced amino acid sequences are similar to corresponding regions of GRK5 and GRK6. Thus, the GRK4 mRNA and the GRK4 protein can exist as four distinct variant forms. The human GRK4 gene is composed of 16 exons extending over 75 kilobase pairs of DNA. The two alternatively spliced exons correspond to exons II and XV. The genomic organization of the GRK4 gene is completely distinct from that of the human GRK2 gene, highlighting the evolutionary distance since the divergence of these two genes. Human GRK4 mRNA is expressed highly only in testis, and both alternative exons are abundant in testis mRNA. The four GRK4 proteins have been expressed, and all incorporate [3H]palmitate. GRK4 is capable of augmenting the desensitization of the rat luteinizing hormone/chorionic gonadotropin receptor upon coexpression in HEK293 cells and of phosphorylating the agonist-occupied, purified beta2-adrenergic receptor, indicating that GRK4 is a functional protein kinase.
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PMID:Characterization of the G protein-coupled receptor kinase GRK4. Identification of four splice variants. 862 39

When the wild type beta1-adrenergic receptor (WT-beta1AR) was expressed in Sf9 cells, the beta1AR-stimulated adenylyl cyclase activities were desensitized by prior treatment with isoproterenol. The extent of beta1AR desensitization was not modified, and the onset was not promoted by the overexpression of G protein-coupled receptor kinase 2 (GRK2), GRK5 or GRK6. However, overexpression of the dominant negative mutant of GRK2 appeared to inhibit desensitization of the beta1AR. The change of the potential protein kinase A phosphorylation site located at the intracellular third loop did not affect beta1AR desensitization. Desensitization of the truncated mutant, in which nearly all of the serine and threonine residues from the carboxyl terminus were eliminated, was the same as that of the WT-beta1AR. A deletion mutant that lacked serine and threonine residues of the intracellular third loop was also desensitized by isoproterenol stimulation. Furthermore, the deletion of serine and threonine residues from both the intracellular third loop and carboxyl terminus did not affect desensitization of the beta1AR. These results suggested that phosphorylation by endogenous GRKs in Sf9 cells contributed to desensitization of the beta1AR and that the regions other than third intracellular loop and carboxyl terminus may be responsible for beta1AR desensitization.
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PMID:Analysis of domain responsible for desensitization of beta1-adrenergic receptor. 1058 Mar 65

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 histamine H2 receptor (H2r) belongs to the heptahelical receptor family; upon agonist binding, members of this family activate a G protein and the downstream effector adenylyl cyclase. Like other G protein-coupled receptors, exposure of H2r to agonists produces a desensitization of the response. The present study focused on the desensitization mechanism of this receptor. Using transiently transfected COS-7 cells expressing tagged-H2r, the desensitization induced by amthamine, characterized by decreased cAMP production, was studied. Results show that the receptor was rapidly desensitized with a t(1/2) = 0.49 +/- 0.01 min. Because of the rapid nature of H2r desensitization, receptor phosphorylation was examined as a likely mechanism for signal attenuation. H2r desensitization was not affected by protein kinases A and C (PKA and PKC) inhibitors but was remarkably reduced by Zn(2+), an inhibitor of G protein-coupled receptor kinases (GRKs). Cotransfection experiments using tagged H2r and different GRKs (2, 3, 5, or 6), demonstrated that GRK2 and GRK3 were the most potent in augmenting desensitization, causing a reduction in the maximal response to amthamine and a decrease of the t(1/2) for desensitization, whereas GRK5 and GRK6 did not affect the signaling. Receptor phosphorylation correlates with desensitization for each GRK studied, whereas phosphorylation that is dependent on protein kinases A and C seemed irrelevant in receptor signal termination. These results indicate that in H2r-transfected COS-7 cells, exposure to an agonist caused desensitization controlled by H2r phosphorylation via GRK2 and GRK3.
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PMID:Histamine H2 receptor desensitization: involvement of a select array of G protein-coupled receptor kinases. 1164 33

Desensitization of G-protein-coupled receptors may involve phosphorylation of serine and threonine residues. The leukotriene B(4) (LTB(4)) receptor (BLT1) contains 14 intracellular serines and threonines, 8 of which are part of consensus target sequences for protein kinase C (PKC) or casein kinase 2. In this study, we investigated the importance of PKC and GPCR-specific kinase (GRK) phosphorylation in BLT1 desensitization. Pretreatment of BLT1-transfected COS-7 cells with PKC activators caused a decrease of LTB(4)-induced inositol phosphate (IP) accumulation. This reduction was prevented with the PKC inhibitor, staurosporine, and not observed in cells expressing a BLT1 deletion mutant (G291stop) lacking the cytoplasmic tail. Moreover LTB(4)-induced IP accumulation was significantly inhibited by overexpression of GRK2, GRK5, and especially GRK6, in cells expressing wild type BLT1 but not in those expressing G291stop. GRK6-mediated desensitization correlated with increased phosphorylation of BLT1. The G319stop truncated BLT1 mutant displayed functional characteristics comparable with wild type BLT1 in terms of desensitization by GRK6, but not by PKC. Substitution of Thr(308) within a putative casein kinase 2 site to proline or alanine in the full-length BLT1 receptor prevented most of GRK6-mediated inhibition of LTB(4)-induced IP production but only partially affected LTB(4)-induced BLT1 phosphorylation. Our findings thus suggest that Thr(308) is a major residue involved in GRK6-mediated desensitization of BLT1 signaling.
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PMID:Threonine 308 within a putative casein kinase 2 site of the cytoplasmic tail of leukotriene B(4) receptor (BLT1) is crucial for ligand-induced, G-protein-coupled receptor-specific kinase 6-mediated desensitization. 1207 28

We previously reported that G protein-coupled receptor kinase (GRK) may contribute to beta-adrenergic receptor (beta-AR) uncoupling occurring just before parturition in rat uterine muscle (myometrium). To identify the GRK involved, we set up in this study a primary cell culture retaining the morphological and functional characteristics of myometrial tissue as well as the in vivo pattern of GRK expression (GRK2, GRK5, and GRK6). In this model, homologous beta-AR desensitization was assessed by an approximately 60% decrease in cAMP production to a subsequent challenge with the beta-agonist, isoproterenol. Desensitization was reduced by 36% with a GRK inhibitor, heparin, and by 31% with a protein kinase A in-hibitor, H89. Using antibodies known to specifically inhibit either GRK2/3 or GRK4-6 families, we demonstrated that only the GRK4-6 family mediated beta-AR desensitization. To discriminate between endogenous GRK5 and GRK6, we attempted to inhibit their action by introducing, into myometrial cells, kinase-dead dominant-negative mutants ((K215R)GRK5 and (K215R)GRK6). Expression of (K215R)GRK6 increased by approximately 70% the cAMP response to isoproterenol without effect on forskolin stimulation. Conversely, expression of (K215R)GRK5 or (K220R)GRK2 had no effect on beta-adrenergic signaling. These results strongly suggest that endogenous GRK6 mediate homologous beta-AR desensitization in myometrial cells.
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PMID:Endogenous G protein-coupled receptor kinase 6 triggers homologous beta-adrenergic receptor desensitization in primary uterine smooth muscle cells. 1281 May 62

Previously we have shown that G protein-coupled receptor kinase (GRK) 6 plays a major role in the regulation of the human M3 muscarinic acetylcholine receptor (M3 mAChR) in the human neuroblastoma SH-SY5Y. However, 30-fold overexpression of the catalytically inactive, dominant-negative K215RGRK6 produced only a 50% suppression of M3 mAChR phosphorylation and desensitization. Here, we have attempted to determine whether other endogenous kinases play a role in the regulation of M3 mAChR signaling. In contrast to the clear attenuating effect of K215RGRK6 expression on M3 mAChR regulation, dominant-negative forms of GRKs (K220RGRK2, K220RGRK3, K215RGRK5) and casein kinase 1alpha (K46RCK1alpha) were without effect. In addition, inhibition of a variety of second-messenger-regulated kinases and the tyrosine kinase Src also had no effect upon agonist-stimulated M3 mAChR regulation. To investigate further the desensitization process we have followed changes in inositol 1,4,5-trisphosphate in single SHSY5Y cells using the pleckstrin homology domain of PLCdelta1 tagged with green fluorescent protein (eGFP-PHPLCdelta1). Stimulation of cells with approximate EC50 concentrations of agonist before and after a desensitizing period of agonist exposure resulted in a marked attenuation of the latter response. Altered GRK6 activity, through overexpression of wild-type GRK6 or K215RGRK6, enhanced or reduced the degree of M3 mAChR desensitization, respectively. Taken together, our data indicate that M3 mAChR desensitization is mediated by GRK6 in human SH-SY5Y cells, and we show that receptor desensitization of phospholipase C signaling can be monitored in 'real-time' in single, living cells.
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PMID:Specificity of g protein-coupled receptor kinase 6-mediated phosphorylation and regulation of single-cell m3 muscarinic acetylcholine receptor signaling. 1457 54


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