Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To investigate the mechanisms of agonist-promoted desensitization of the alpha 2-adrenergic receptor (alpha 2AR), the human alpha 2AAR and a mutated form of the receptor were expressed in CHW cells. After cells were exposed to epinephrine for 30 min, the ability of the wild type alpha 2AAR to mediate inhibition of forskolin-stimulated adenylyl cyclase was depressed by approximately 78%. To assess the role of receptor phosphorylation during desensitization, cells were incubated with 32Pi, exposed to agonist, and alpha 2AAR purified by immunoprecipitation with a fusion protein antibody. Agonist-promoted desensitization was found to be accompanied by phosphorylation of the alpha 2AAR in vivo. The beta-adrenergic receptor kinase (beta ARK) is known to phosphorylate purified alpha 2AAR in vitro. We found that heparin, a beta ARK inhibitor, ablated short term agonist-induced desensitization of alpha 2AAR, while such desensitization was unaffected by inhibition of protein kinase A. To further assess the role of beta ARK, we constructed a mutated alpha 2AAR which has a portion of the third intracellular loop containing 9 serines and threonines (potential phosphorylation sites) deleted. This mutated alpha 2AAR failed to undergo short term agonist-induced desensitization. Agonist promoted in vivo phosphorylation of this mutated receptor was reduced by 90%, consistent with the notion that receptor phosphorylation at sites in the third intracellular loop plays a critical role in alpha 2AAR desensitization. After 24 h of agonist exposure, an even more profound desensitization of alpha 2AAR occurred, which was not accompanied by a decrease in receptor expression. Rather, long term agonist-induced desensitization was found to be due in part to a decrease in the amount of cellular Gi, which was not dependent on receptor third loop phosphorylation sites.
 ...
PMID:Sites in the third intracellular loop of the alpha 2A-adrenergic receptor confer short term agonist-promoted desensitization. Evidence for a receptor kinase-mediated mechanism. 131 18

Exposure of mouse colliculi neurons to selective 5-hydroxytryptamine (5-HT)4 agonists was accompanied by a rapid desensitization of the receptor-stimulated adenylyl cyclase response. Half-maximal desensitization occurred after 2 min. Only exposure of neurons to selective 5-HT4 agonists led to a potent desensitization of the 5-HT4-mediated response. Neurons exposed to other agents, like isoproterenol, vasoactive intestinal peptide, or forskolin, that increase cAMP levels did not undergo any desensitization of 5-HT4 receptors. Activation of protein kinase A with either 8-bromo-cAMP or dibutyryl-cAMP or application of inhibitors of protein kinase A-dependent phosphorylation did not change the rate of 5-HT4-induced desensitization. No shift to lower potency of 5-HT4 agonists in the concentration-response curve was observed. These results suggest that 5-HT4 receptor agonists induced homologous but not cAMP-mediated heterologous desensitization. A good correlation was found between the affinities of nine 5-HT4 agonists and their abilities to desensitize the adenylyl cyclase response. This may indicate that homologous desensitization is a function of the mean occupancy time of the receptors by agonists. When permeabilized neurons were loaded with heparin, an inhibitor of the beta-adrenergic receptor kinase (beta ARK), 5-HT4 receptor desensitization was reduced by 30-40%. Interestingly, Zn2+, an other inhibitor of beta ARK, totally prevented 5-HT4-induced desensitization. Pretreatment of neurons with concanavalin A, reported to inhibit sequestration of beta-adrenergic receptors from the cell surface, reduced the desensitization process by 70%. These data suggest that both sequestration and phosphorylation by beta ARK, or another specific agonist-dependent receptor kinase, are involved in homologous desensitization of 5-HT4 receptors coupled to adenylyl cyclase.
 ...
PMID:Characterization of homologous 5-hydroxytryptamine4 receptor desensitization in colliculi neurons. 133 63

Homologous desensitization of beta-adrenergic receptors, as well as adaptation of rhodopsin, are thought to be triggered by specific phosphorylation of the receptor proteins. However, phosphorylation alone seems insufficient to inhibit receptor function, and it has been proposed that the inhibition is mediated, following receptor phosphorylation, by the additional proteins beta-arrestin in the case of beta-adrenergic receptors and arrestin in the case of rhodopsin. In order to test this hypothesis with isolated proteins, beta-arrestin and arrestin were produced by transient overexpression of their cDNAs in COS7 cells and purified to apparent homogeneity. Their functional effects were assessed in reconstituted receptor/G protein systems using either beta 2-adrenergic receptors with Gs or rhodopsin with Gt. Prior to the assays, beta 2-receptors and rhodopsin were phosphorylated by their specific kinases beta-adrenergic receptor kinase (beta ARK) and rhodopsin kinase, respectively. beta-Arrestin was a potent inhibitor of the function of beta ARK-phosphorylated beta 2-receptors. Half-maximal inhibition occurred at a beta-arrestin:beta 2-receptor stoichiometry of about 1:1. More than 100-fold higher concentrations of arrestin were required to inhibit beta 2-receptor function. Conversely, arrestin caused half-maximal inhibition of the function of rhodopsin kinase-phosphorylated rhodopsin when present in concentrations about equal to those of rhodopsin, whereas beta-arrestin at 100-fold higher concentrations had little inhibitory effect. The potency of beta-arrestin in inhibiting beta 2-receptor function was increased over 10-fold following phosphorylation of the receptors by beta ARK, but was not affected by receptor phosphorylation using protein kinase A. This suggests that beta-arrestin plays a role in beta ARK-mediated homologous, but not in protein kinase A-mediated heterologous desensitization of beta-adrenergic receptors. It is concluded that even though arrestin and beta-arrestin are similar proteins, they display marked specificity for their respective receptors and that phosphorylation of the receptors by the receptor-specific kinases serves to permit the inhibitory effects of the "arresting" proteins by allowing them to bind to the receptors and thereby inhibit their signaling properties. Furthermore, it is shown that this mechanism of receptor inhibition can be reproduced with isolated purified proteins.
 ...
PMID:Receptor-specific desensitization with purified proteins. Kinase dependence and receptor specificity of beta-arrestin and arrestin in the beta 2-adrenergic receptor and rhodopsin systems. 134 18

The beta-adrenergic receptor kinase (beta-ARK) phosphorylates G protein coupled receptors in an agonist-dependent manner. Since the exact sites of receptor phosphorylation by beta-ARK are poorly defined, the identification of substrate amino acids that are critical to phosphorylation by the kinase are also unknown. In this study, a peptide whose sequence is present in a portion of the third intracellular loop region of the human platelet alpha 2-adrenergic receptor is shown to serve as a substrate for beta-ARK. Removal of the negatively charged amino acids surrounding a cluster of serines in this alpha 2-peptide resulted in a complete loss of phosphorylation by the kinase. A family of peptides was synthesized to further study the role of acidic amino acids in peptide substrates of beta-ARK. By kinetic analyses of the phosphorylation reactions, beta-ARK exhibited a marked preference for negatively charged amino acids localized to the NH2-terminal side of a serine or threonine residue. While there were no significant differences between glutamic and aspartic acid residues, serine-containing peptides were 4-fold better substrates than threonine. Comparing a variety of kinases, only rhodopsin kinase and casein kinase II exhibited significant phosphorylation of the acidic peptides. Unlike beta-ARK, RK preferred acid residues localized to the carboxyl-terminal side of the serine. A feature common to beta-ARK and RK was a much greater Km for peptide substrates as compared to that for intact receptor substrates.
 ...
PMID:Role of acidic amino acids in peptide substrates of the beta-adrenergic receptor kinase and rhodopsin kinase. 164 91

Three separate processes may contribute to rapid beta-adrenergic receptor desensitization: functional uncoupling from the stimulatory guanine nucleotide-binding protein Gs, mediated by phosphorylation of the receptors by two distinct kinases, the specific beta-adrenergic receptor kinase (beta ARK) and the cyclic AMP-dependent protein kinase A (PKA), as well as a spatial uncoupling via sequestration of the receptors away from the cell surface. To evaluate the relative importance and potential role of the various processes in different physiological situations, a kinetic analysis of these three mechanisms was performed in permeabilized A431 epidermoid carcinoma cells. To allow a separate analysis of each mechanism, inhibitors of the various desensitization mechanisms were used: heparin to inhibit beta ARK, the PKA inhibitor peptide PKI to inhibit PKA, and concanavalin A treatment to prevent sequestration. Isoproterenol-induced phosphorylation of beta 2 receptors in these cells by beta ARK occurred with a t1/2 of less than 20 sec, whereas phosphorylation by PKA had a t1/2 of about 2 min. Similarly, beta ARK-mediated desensitization of the receptors proceeded with a t1/2 of less than 15 sec, and PKA-mediated desensitization with a t1/2 of about 3.5 min. Maximal desensitization mediated by the two kinases corresponded to a reduction of the signal-transduction capacity of the receptor/adenylyl cyclase system by about 60% in the case of beta ARK and by about 40% in the case of PKA. Receptor sequestration was much slower (t1/2 of about 10 min) and involved no more than 30% of the cell surface receptors. It is concluded that beta ARK-mediated phosphorylation is the most rapid and quantitatively most important factor contributing to the rapid desensitization. This rapidity of the beta ARK-mediated mechanism makes it particularly well suited to regulate beta-adrenergic receptor function in rapidly changing environments such as the synaptic cleft.
 ...
PMID:Comparative rates of desensitization of beta-adrenergic receptors by the beta-adrenergic receptor kinase and the cyclic AMP-dependent protein kinase. 164 31

Light-dependent deactivation of rhodopsin as well as homologous desensitization of beta-adrenergic receptors involves receptor phosphorylation that is mediated by the highly specific protein kinases rhodopsin kinase (RK) and beta-adrenergic receptor kinase (beta ARK), respectively. We report here the cloning of a complementary DNA for RK. The deduced amino acid sequence shows a high degree of homology to beta ARK. In a phylogenetic tree constructed by comparing the catalytic domains of several protein kinases, RK and beta ARK are located on a branch close to, but separate from the cyclic nucleotide-dependent protein kinase and protein kinase C subfamilies. From the common structural features we conclude that both RK and beta ARK are members of a newly delineated gene family of guanine nucleotide-binding protein (G protein)-coupled receptor kinases that may function in diverse pathways to regulate the function of such receptors.
 ...
PMID:The receptor kinase family: primary structure of rhodopsin kinase reveals similarities to the beta-adrenergic receptor kinase. 165 54

We have partially purified a protein kinase that phosphorylates muscarinic receptors (mAChR) in the presence of agonists and have shown that the phosphorylation is stimulated by the beta gamma subunits of the GTP binding protein Go (Haga, K., and Haga, T. (1990) FEBS Lett. 268, 43-47). We report here that rhodopsin is also phosphorylated in a light-dependent manner by the same kinase preparation and that beta gamma subunits derived from Gs, Gi, and Go stimulate the phosphorylation of both rhodopsin and mAChRs. The rhodopsin- and mAChR-phosphorylating activities were eluted in the same fractions using a purification procedure that is essentially the same as that used for the purification of beta-adrenergic receptor kinase (Benovic, J.L., Strasser, R.H., Caron, M.G., and Lefkowitz, R.J. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2797-2801) and were inhibited by low concentrations of heparin, an inhibitor of beta-adrenergic receptor kinase, (IC50 = 15 nM), suggesting that both mAChR and rhodopsin are phosphorylated by the same or very similar kinase(s) belonging to the beta-adrenergic receptor kinase family. G protein beta gamma subunits increased the Vmax of the phosphorylation of rhodopsin 12-fold. Kinetic data were consistent with the assumptions that the protein kinase (mAChR kinase) binds rhodopsin and beta gamma subunits in a random order and that the reaction rate is proportional to concentration of the ternary complex. By contrast, the light-dependent phosphorylation of rhodopsin by the rhodopsin kinase was not stimulated by the beta gamma subunits. These results indicate that beta gamma subunits may interact with and activate the mAChR kinase but not rhodopsin kinase and suggest that the beta gamma subunit of G proteins may take part in the desensitization of G protein-linked receptors.
 ...
PMID:Activation by G protein beta gamma subunits of agonist- or light-dependent phosphorylation of muscarinic acetylcholine receptors and rhodopsin. 173 28

The integrity of coupling of the beta 2-adrenergic receptor (beta 2AR) to its guanine nucleotide-binding protein, Gs, and phosphorylation events on the receptor molecule have been proposed to be important determinants in the processes of receptor sequestration and down-regulation. However, little is known about the molecular mechanisms underlying these processes, and the regions of the receptor molecule that specifically subserve sequestration and down-regulation have yet to be delineated. To address these questions, we stably transfected eight mutant beta 2AR genes into Chinese hamster fibroblasts and evaluated the coupling, sequestration, and down-regulation properties of the mutated receptors. These mutant receptors have been previously demonstrated either to exhibit abnormal coupling to Gs or to lack functionally important phosphorylation sites for either the cAMP-dependent protein kinase or the agonist-dependent beta-adrenergic receptor kinase. All eight mutants exhibited receptor sequestration equivalent in extent to that of the beta 2AR, regardless of their coupling or phosphorylation status. However, four mutants that exhibited various degrees of impairment in coupling to Gs showed blunted receptor down-regulation patterns. Simultaneous treatment with isoproterenol and dibutyryl-cAMP did not improve the abilities of the mutant receptors to undergo down-regulation. These findings demonstrate that a variety of mutant beta 2AR with impaired coupling to Gs are, nevertheless, able to be sequestered normally. In contrast, agonist-induced down-regulation appears to require coupling of the beta 2AR to Gs but is largely independent of the generation of cAMP. Our results also suggest that molecular determinants of the beta 2AR involved in receptor sequestration are distinct from those participating in the down-regulation process.
 ...
PMID:Mutations of the human beta 2-adrenergic receptor that impair coupling to Gs interfere with receptor down-regulation but not sequestration. 184 93

Plasma membrane receptors that couple to guanine nucleotide-binding regulatory proteins (G proteins) undergo a variety of rapid (minutes) and longer term (hours) regulatory processes induced by ligands. For the beta 2-adrenergic receptor (beta 2AR), the rapid processes include functional desensitization, mediated by phosphorylation of the receptor by the cAMP-dependent protein kinase and the beta-adrenergic receptor kinase, as well as a loss of hydrophilic ligand binding proposed to represent sequestration of receptors into a cellular compartment distinct from the plasma membrane. The slower processes include beta 2AR down-regulation (i.e., a decrease in the total cellular complement of receptors). It is not yet known whether beta 2AR phosphorylation and/or sequestration are prerequisites for down-regulation of the receptor. Like other G protein-coupled receptors, the beta 2AR molecule spans the plasma membrane seven times, and the cytoplasmic carboxyl-terminal domain has been proposed to contain molecular determinants for each of these regulatory processes. We replaced four serine and threonine residues located within a 10-amino acid segment of this domain of beta 2AR and thereby prevented agonist-promoted phosphorylation, sequestration, and rapid desensitization of the adenylyl cyclase response. In contrast, these mutations did not affect functional coupling to the stimulatory G protein Gs or long-term down-regulation. These findings thus define a small, hitherto unappreciated region of the receptor molecule that may selectively subserve its rapid regulation. In addition, with the demonstration that beta 2AR does not have to be phosphorylated or sequestered in order to enter the down-regulation pathway, the results suggest that the classical receptor endocytosis model may not be appropriate for beta 2AR regulation.
 ...
PMID:A small region of the beta-adrenergic receptor is selectively involved in its rapid regulation. 184 41

Human SK-N-MC neurotumor cells express beta 1- but not beta 2-adrenergic receptors. Following exposure of the cells to isoproterenol, there was no reduction in the maximum response of adenylyl cyclase to the agonist but a 3-fold shift to less sensitivity in the concentration response. This desensitization was very rapid and dose dependent; half-maximal effects occurred at 10 nM isoproterenol. A similar shift was observed when membranes from control cells were incubated with ATP and the catalytic subunit of cyclic AMP-dependent protein kinase (PKA). No shift, however, was observed in intact cells exposed to either dibutyryl cyclic AMP or dopamine, which stimulates adenylyl cyclase in these cells through D1 dopamine receptors. To pursue the role of protein kinases in the desensitization process, cells were made permeable, loaded with a PKA inhibitor or with heparin, an inhibitor of the beta-adrenergic receptor kinase (beta ARK), and exposed to isoproterenol. The PKA inhibitor but not heparin blocked the agonist-mediated desensitization. In contrast, desensitized human tumor cells (HeLa and A431), which express beta 2-adrenergic receptors, exhibited both a shift in concentration response and a reduction in maximum response; the former was blocked by the PKA inhibitor and the latter by heparin. Our results indicated that whereas both human beta 1- and beta 2-adrenergic receptors are susceptible to PKA, only the beta 2 receptors are susceptible to beta ARK. These differences in desensitization may be due to differences in receptor structure as the human beta 1 receptor has fewer potential phosphorylation sites for beta ARK in the carboxyl terminus than the human beta 2 receptor.
 ...
PMID:Desensitization of the human beta 1-adrenergic receptor. Involvement of the cyclic AMP-dependent but not a receptor-specific protein kinase. 185 Apr 9


1 2 3 4 5 6 Next >>