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)

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

Using a clonal cell line that stably expresses the murine luteinizing hormone receptor (LHR 11/6 cells), we studied the molecular mechanisms of agonist-induced desensitization of the luteinizing hormone/chorionic gonadotropin-responsive adenylyl cyclase. Exposure of transfected cells to human chorionic gonadotropin (hCG) resulted in a dose-dependent loss of maximal hCG-stimulable adenylyl cyclase activity without a significant shift to the right of the dose-response curve to hCG. This rapid uncoupling of the LH receptor from the cellular adenylyl cyclase system was not accompanied by internalization of receptor sites. A 6-h exposure to hCG led only to minor (ca. 25%) loss of membrane binding sites. The dose-response curve to hCG was not altered by pretreating cells with 8-Br-cAMP or prostaglandin E1. These findings, and the observation that hCG-induced desensitization can still be monitored at Mg2+ concentrations in the assay as high as 10 mM, preclude a significant contribution of protein kinase A to LH receptor uncoupling. The murine LH receptor not only stimulates adenylyl cyclase but also phospholipase C and probably protein kinase C (PKC) via diacylglycerol. Activation of PKC by 4 beta-phorbol 12-myristate 13-acetate failed to desensitize. When PKC was down-regulated hCG could still exert a maximal desensitizing effect. It is concluded that in LHR 11/6 cells there is no evidence for a major role of PKC in homologous desensitization. Thus, it is likely that a second messenger-independent kinase, such as beta-adrenergic receptor kinase, or a different, as yet unknown mechanism is involved in the agonist-induced desensitization of the LH receptor.
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PMID:Homologous desensitization of the murine luteinizing hormone receptor expressed in L cells. 767 43

Guanine nucleotide binding protein (G-protein)-coupled receptor kinases (GRKs) specifically phosphorylate the agonist-occupied form of G-protein-coupled receptors such as the beta 2-adrenergic receptor and rhodopsin. The best characterized members of this family include the beta-adrenergic receptor kinase (beta ARK) and rhodopsin kinase. To identify additional members of the GRK family, the polymerase chain reaction was used to amplify human heart cDNA using degenerate oligonucleotide primers from highly conserved regions unique to the GRK family. Here we report the isolation of a cDNA that encodes a 590-amino acid protein kinase, termed GRK5, which has 34.8% and 47.2% amino acid identities with beta ARK and rhodopsin kinase, respectively. Interestingly, GRK5 has an even higher homology with Drosophila GPRK-2 (71.0% identity) and the recently identified human IT11 (69.1% identity). Northern blot analysis of GRK5 with selected human tissues reveals a message of approximately 3 kilobases with highest levels in heart, placenta, lung > skeletal muscle > brain, liver, pancreas > kidney. GRK5, overexpressed in Sf9 insect cells using the baculovirus system, was able to phosphorylate rhodopsin in a light-dependent manner. In addition, GRK5 neither contains a consensus sequence for isoprenylation like rhodopsin kinase nor is activated by G-protein beta gamma subunits like beta ARK1. Thus, GRK5 represents a member of the GRK family that likely has a unique physiological role.
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PMID:Cloning and expression of GRK5: a member of the G protein-coupled receptor kinase family. 768 6

Raf-1 is a serine/threonine protein kinase positioned downstream of Ras in the mitogen-activated protein kinase cascade. Using a yeast two-hybrid strategy to identify other proteins that interact with and potentially regulate Raf-1, we isolated a clone encoding the carboxyl-terminal half of the G beta 2 subunit of heterotrimeric G-proteins. In vitro, purified G beta gamma subunits specifically bound to a GST fusion protein encoding amino acids 1-330 of Raf-1 (Raf/330). Binding assays with truncation mutants of GST-Raf indicate that the region located between amino acids 136 and 239 is a primary determinant for interaction with G beta gamma. In competition experiments, the carboxyl terminus of beta-adrenergic receptor kinase (beta ARK) blocked the binding of G beta gamma to Raf/330; however, the Raf-1-binding proteins, Ras and 14-3-3, had no effect. Scatchard analysis of in vitro binding between Raf/330 and G beta gamma revealed an affinity of interaction (Kd = 163 +/- 36 nM), similar to that seen between G beta gamma and beta ARK (Kd = 87 +/- 24 nM). The formation of native heterotrimeric G alpha beta gamma complexes, as measured by pertussis toxin ADP-ribosylation of G alpha, could be disrupted by increasing amounts of Raf/330, with an EC50 of approximately 200 nM, in close agreement with the estimated binding affinity. In vivo complexes of Raf-1 and G beta gamma were isolated from human embryonic kidney 293-T cells transfected with epitope-tagged G beta 2. The identification and characterization of this novel interaction raises several possibilities for signaling cross-talk between growth factor receptors and those receptors coupled to heterotrimeric G-proteins.
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PMID:A direct interaction between G-protein beta gamma subunits and the Raf-1 protein kinase. 778 77

Consequent to agonist exposure, many G protein-coupled receptors undergo sequestration or internalization. Results with receptors linked to adenylate cyclase, such as the beta 2-adrenergic receptor, or receptors linked to phospholipase C (PLC) have provided conflicting results regarding the role of second messenger-dependent (i.e., protein kinase A or C) and -independent (i.e., beta-adrenergic receptor kinase) kinases in mediating this process. Recent results for truncated and mutated gastrin-releasing peptide (GRP) receptors (GRP-R), as well as muscarinic cholinergic receptors, suggest that activation of protein kinase C may be needed for full receptor internalization. Nearly all G protein-coupled receptors studied to date, including the GRP-R, possess two highly conserved amino acids that are important in mediating receptor-G protein coupling to second messengers, i.e., arginine in the proximal second intracellular loop and alanine in the distal third intracellular loop. We selectively mutated each of these residues in the GRP-R to determine their importance for activation of PLC. Site-directed mutagenesis was performed to change arginine at position 139 to glycine (R139G mutant) and alanine at position 263 to glutamate (A263E mutant), with stable cell lines being created by transfection of the wild-type or mutated receptor cDNA into BALB/3T3 fibroblasts. Both R139G (Kd = 12.0 +/- 1.6 nM) and A263E (Kd = 12.2 +/- 1.7 nM) had a lower affinity for bombesin than did wild-type GRP-R (Kd = 1.4 +/- 0.4 nM); however, characteristic stoichiometries for the binding of agonists to this receptor were maintained equally in all three cell lines (bombesin > GRP >> neuromedin B). The wild-type GRP-R exposed to bombesin increased [3H]inositol phosphates (a measure of PLC activation) approximately 4-fold, with an EC50 of 5.1 +/- 2.2 nM. In contrast, [3H]inositol phosphates were not significantly increased in cells expressing R139G or A263E receptors, demonstrating that Arg139 and Ala263 are required for GRP-R activation of PLC. However, when receptor internalization at 37 degrees was assessed by ligand acid-stripping studies, 53 +/- 2% of A263E receptors were internalized at 90 min, compared with 85 +/- 5% of wild-type GRP-R, whereas only 10 +/- 3% of R139G receptors were internalized. Preincubation of either mutant cell line with 100 nM 12-O-tetradecanoylphorbol-13-acetate markedly increased internalization rates, such that at 90 min 62 +/- 2% of R139G receptors and 82 +/- 1% of A263E receptors were internalized.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Internalization of the gastrin-releasing peptide receptor is mediated by both phospholipase C-dependent and -independent processes. 793 30

Although a role for the beta gamma-subunits of heterotrimeric G proteins (G beta gamma) in signal transduction by several cellular systems has been established, the structural features of cellular proteins interacting with G beta gamma have yet to be fully elucidated. The G beta gamma-binding region of beta-adrenergic receptor kinase (beta ARK), a cytosolic enzyme recruited to the membrane receptor substrate by G beta gamma, has been localized to the carboxyl terminus of the enzyme. Here, we demonstrate that the amino terminus of phosducin, a 33-kDa G beta gamma-binding retinal phosphoprotein, contains sequences homologous with the G beta gamma-binding domain of beta ARK. Accordingly, a glutathione S-transferase-fusion protein containing only the amino-terminal 105 amino acids of phosducin displayed G beta gamma binding ability. This domain of phosducin contains a protein kinase A (PKA) phosphorylation site, and upon phosphorylation, the binding of full-length phosducin to G beta gamma is reduced. In addition, transient expression of phosducin in COS-7 cells significantly inhibits G beta gamma-mediated phosphoinositide hydrolysis. This inhibitory effect is completely reversed by pretreatment of cells with dibutyryl cAMP, an activator of PKA. Thus, the binding of G beta gamma to phosducin can be regulated by PKA-phosphorylation in an intact cell model system.
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PMID:Determination of the G beta gamma-binding domain of phosducin. A regulatable modulator of G beta gamma signaling. 796 75

The roles of three protein kinases, cyclic AMP-dependent protein kinase (protein kinase A), protein kinase C, and beta-adrenergic receptor kinase (beta ARK), implicated in agonist-induced desensitization of guanine nucleotide-binding protein (G-protein)-coupled receptors were explored in four different cell lines after 48 hr of incubation with oligodeoxynucleotides antisense to the mRNA encoding each kinase. Desensitization of beta 2-adrenergic receptors was analyzed in cell types in which the activities of the endogenous complement of protein kinases A and C and beta ARK were distinctly different. Protein kinase A was necessary for desensitization of rat osteosarcoma cells (ROS 17/2.8), whereas the contribution of beta ARK to desensitization was insignificant. In Chinese hamster ovary cells that stably express beta 2-adrenergic receptors and in smooth muscle cells (DDT1MF-2), oligodeoxynucleotides antisense to beta ARK mRNA nearly abolished desensitization, whereas oligodeoxynucleotides antisense to protein kinase A mRNA attenuated desensitization to a lesser extent. In human epidermoid carcinoma cells (A-431), oligodeoxynucleotides antisense to either protein kinase A mRNA or beta ARK mRNA attenuated agonist-induced desensitization, providing a third scenario in which two kinases constitute the basis for agonist-induced desensitization. In sharp contrast, oligodeoxynucleotides antisense to protein kinase C mRNA were found to enhance rather than attenuate desensitization in DDT1MF-2 and A-431 cell lines, demonstrating counterregulation between prominent protein kinases in desensitization. Using antisense oligodeoxynucleotides to "knock out" target protein kinases in vivo, we reveal distinctive cell-type-specific roles of protein kinase A, protein kinase C, and beta ARK in agonist-induced desensitization.
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PMID:Oligodeoxynucleotides antisense to mRNA encoding protein kinase A, protein kinase C, and beta-adrenergic receptor kinase reveal distinctive cell-type-specific roles in agonist-induced desensitization. 799 5

Exposure of beta-adrenergic receptors (BAR) to agonists often leads to a rapid loss of receptor responsiveness. The proposed mechanisms of such rapid receptor desensitization include receptor phosphorylation by either cAMP-dependent protein kinase or the specific beta-adrenergic receptor kinase (BARK), leading to functional uncoupling from adenylyl cyclase and sequestration of the receptors away from the cell surface. To evaluate the physiological role of such mechanisms, we have investigated whether rapid regulation of BAR occurs in the neonatal rat liver immediately after birth, a physiological situation characterized by a dramatic but transient increase in plasma catecholamines. We have detected a rapid, transient uncoupling of liver plasma membrane BARs from adenylyl cyclase (corresponding to a desensitization of approximately 45%) within the first minutes of extrauterine life, followed by a transient sequestration of approximately 40% of the BARs away from the plasma membrane. In agreement with such pattern of desensitization, we have detected (by enzymatic and immunological assays) rapid changes in BARK specific activity in different neonatal rat liver subcellular fractions that take place within the same time frame of BAR uncoupling and sequestration. Our results provide new evidence on the potential role of BAR desensitization mechanisms in vivo and suggest that they are involved in modulating catecholamines actions at the moment of birth. Furthermore, our data indicate that in addition to its suggested role as a rapid modulator of adrenergic receptor function at synapse, rapid BARK-mediated receptor regulation may have functional relevance in other tissues in response to high circulating or local levels of agonists.
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PMID:Rapid desensitization of neonatal rat liver beta-adrenergic receptors. A role for beta-adrenergic receptor kinase. 813 79

Because the acute homologous phase of desensitization of the LH/CG-sensitive adenylyl cyclase in porcine follicles is readily demonstrated in a cell-free membrane preparation, it follows that any enzyme(s) required to achieve desensitization must be present in the membranes and must be activated upon LH/CG receptor activation. The purpose of the following studies was to determine whether modulation of endogenous membrane protein kinases, with activators or inhibitors, or addition of exogenous protein kinases affected desensitization of the LH/CG-sensitive adenylyl cyclase. The effects of these potential modulators were evaluated in both the presence and absence of ligand (hCG)-stimulated receptor activation. To this end, membranes were incubated in the presence or absence of hCG (stage 1) and then assayed for adenylyl cyclase activity in the presence or absence of hCG (stage 2). The results showed that although porcine follicular membranes rich in LH/CG-sensitive adenylyl cyclase activity also exhibited cAMP-dependent [protein kinase-A (PKA)], cGMP-dependent (PKG), lipid-dependent (PKC), Ca2+/calmodulin, and casein kinase-I and -II activities, only full hCG-stimulated adenylyl cyclase activity (measured with BSA in stage 1 and hCG in stage 2) was reduced upon addition of exogenous PKC (to the stage 1 incubation). hCG-dependent desensitization of cAMP synthesis (measured with hCG in stages 1 and 2) was unaffected by activators or inhibitors of endogenous PKA, PKC, or PKG, by an inhibitor of casein kinases and kinases in the beta-adrenergic receptor kinase family, or by the addition of exogenous active PKA, PKC, or rhodopsin kinase to the stage 1 incubation. These results suggest that the acute homologous phase of hCG-dependent desensitization of adenylyl cyclase activity in follicular membranes is not regulated by PKA, PKC, PKG, or messenger-independent heparin-sensitive protein kinases.
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PMID:The effect of protein kinases on desensitization of the porcine follicular membrane luteinizing hormone/chorionic gonadotropin-sensitive adenylyl cyclase. 813 39

The beta-adrenergic receptor kinase (beta ARK) specifically phosphorylates the activated form of the beta 2-adrenergic receptor (beta 2AR) and related G protein-coupled receptors. To further elucidate the role of beta ARK in receptor desensitization, we generated a beta ARK dominant negative mutant by converting an invariant lysine residue in the protein kinase catalytic domain to an arginine. Expressed and purified beta ARK-K220R was able to inhibit wild type beta ARK phosphorylation of the beta 2AR in vitro. When stably transfected into human bronchial epithelial BEAS-2B cells, beta ARK-K220R promoted a > 2-fold increase in beta-agonist-stimulated cAMP production without affecting beta 2AR sequestration. In contrast, beta ARK-K220R had no effect on the desensitization of the prostaglandin E2 receptor response in BEAS-2B cells. These findings directly demonstrate a role for beta ARK in desensitization of the beta 2AR in intact cells and establish the potential utility of using dominant negative mutants to elucidate the substrate specificity of G protein-coupled receptor kinases.
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PMID:A beta-adrenergic receptor kinase dominant negative mutant attenuates desensitization of the beta 2-adrenergic receptor. 817 32


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