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)

Exposure of beta 2-adrenergic receptors (beta 2ARs) to agonists causes a rapid desensitization of the receptor-stimulated adenylyl cyclase response. Phosphorylation of the beta 2AR by several distinct kinases plays an important role in this desensitization phenomenon. In this study, we have utilized purified hamster lung beta 2AR and stimulatory guanine nucleotide binding regulatory protein (Gs), reconstituted in phospholipid vesicles, to investigate the molecular properties of this desensitization response. Purified hamster beta 2AR was phosphorylated by cAMP-dependent protein kinase (PKA), protein kinase C (PKC), or beta AR kinase (beta ARK), and receptor function was determined by measuring the beta 2AR-agonist-promoted Gs-associated GTPase activity. At physiological concentrations of Mg2+ (less than 1 mM), receptor phosphorylation inhibited coupling to Gs by 60% (PKA), 40% (PKC), and 30% (beta ARK). The desensitizing effect of phosphorylation was, however, greatly diminished when assays were performed at concentrations of Mg2+ sufficient to promote receptor-independent activation of Gs (greater than 5 mM). Addition of retinal arrestin, the light transduction component involved in the attenuation of rhodopsin function, did not enhance the uncoupling effect of beta ARK phosphorylation of beta 2AR when assayed in the presence of 0.3 mM free Mg2+. At concentrations of Mg2+ ranging between 0.5 and 5.0 mM, however, significant potentiation of beta ARK-mediated desensitization was observed upon arrestin addition. At a free Mg2+ concentration of 5 mM, arrestin did not potentiate the inhibition of receptor function observed on PKA or PKC phosphorylation. These results suggest that distinct pathways of desensitization exist for the receptor phosphorylated either by PKA or PKC or alternatively by beta ARK.
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PMID:Desensitization of the isolated beta 2-adrenergic receptor by beta-adrenergic receptor kinase, cAMP-dependent protein kinase, and protein kinase C occurs via distinct molecular mechanisms. 134 86

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.
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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

1. The function of trout RBC Na+/H+ antiport is unrelated to cell volume or cell pH regulation. Its role is to improve oxygen transport capacity when the supply of oxygen becomes limited. 2. Antiport activation, mediated by cAMP, promotes complex changes in blood pH which have been analyzed in vivo and in vitro. 3. The regulation of antiport (activation, desensitization, control by molecular oxygen and by a newly discovered cytosolic protein, arrestin) is presented. 4. Molecular cloning of the antiport shows that two typical site motifs of phosphorylation by cAMP-dependent protein kinase are localized on the cytoplasmic region.
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PMID:Regulation of Na+/H+ exchange and pH in erythrocytes of fish. 135 21

Absorption of a photon of light by rhodopsin triggers mechanisms responsible for excitation as well as regulation of the phototransduction cascade. Arrestins are a family of proteins that appear to be responsible for terminating the active state of G-protein-coupled receptors. One of the major substrates of light-dependent phosphorylation in the visual cascade of Drosophila was purified and partially sequenced. The complete primary structure of the protein was determined by isolating the corresponding gene, which revealed it to be a new isoform of arrestin, Arr2. Arr2 is 401 residues in length, and shares 47% sequence identity with the Drosophila Arr1 protein and 42% with human arrestin. We show that the two Drosophila arrestin genes are differentially regulated, and that Arr2 is a specific substrate for a calcium-dependent protein kinase. This is the first demonstration of in vivo regulation of arrestins in a transduction cascade, and provides a new level of modulation in the function of G-protein-coupled receptors.
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PMID:Isolation of a novel visual-system-specific arrestin: an in vivo substrate for light-dependent phosphorylation. 212 11

Photoactivated rhodopsin (R) catalyses, by repetitively interacting with many copies of a guanosine nucleotide binding protein (transducin), the amplified binding of GTP to transducin molecules which then activate cyclic GMP phosphodiesterase. Electrophysiologists recently have shown that cyclic GMP keeps ion channels in the plasma membrane of the rod outer segment open in darkness, and that light-induced hydrolysis of cyclic GMP leads to closure of the channels and therefore to hyperpolarization of the rod cell. Photoactivated rhodopsin interacts not only with transducin, but with two more proteins: a protein kinase that specifically phosphorylates R (in contrast to dark-adapted rhodopsin) at multiple sites; and an abundant soluble protein of 48 KDal (called 48 K-protein, S-antigen, or arrestin) that specifically binds to phosphorylated R. Phosphorylation partially suppresses the ability of R to catalyze transducin-mediated phosphodiesterase activation even in the absence of arrestin. Binding of arrestin to the phosphorylated R potentiates this inhibitory effect, most probably because arrestin competes with transducin for binding on the phosphorylated R. Phosphorylation, in conjunction with arrestin binding, therefore appears to be a mechanism that terminates the active state of the receptor, R.
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PMID:Deactivation of photoactivated rhodopsin by rhodopsin-kinase and arrestin. 304 Sep 78

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

In the retinas of teleost fish dopamine, released from interplexiform cells, modulates synaptic transmission at both the chemical and electrical synapses of retinal horizontal cells. This modulation is due to activation of adenylate cyclase and phosphorylation by protein kinase A, perhaps of the synaptic ion channel proteins themselves. In this study we have fractionated the white perch retina by Percoll density gradient centrifugation in order to identify proteins which coenrich with horizontal cells. In addition we have tested retinal fractions for phosphorylation by native cAMP-dependent kinase. Our findings indicate that there are at least 3 proteins of molecular weights 28, 43/44 and 50 kDa which coenrich with horizontal cells and 3 proteins of 30/31 kDa, 35 kDa (putative rhodopsin) and 48 kDa (putative arrestin) which coenrich with photoreceptor fractions. The 43/44 kDa phosphoprotein is a target for cAMP-dependent protein phosphorylation and thus is apparently an element of the dopaminergic modulatory pathway in perch horizontal cells.
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PMID:Protein content and cAMP-dependent phosphorylation of fractionated white perch retina. 782 Jun 51

Calcium (Ca2+) plays an integral role in the light response of the photoreceptors in both vertebrate and invertebrate organisms. In the ventral eye of the horseshoe crab, Limulus polyphemus, a flash of light delivered to a dark-adapted photoreceptor stimulates a rapid rise in intracellular free calcium concentration ([Ca2+]i), which in turn mediates light adaptation. It has previously been demonstrated that in Limulus photoreceptors light, via Ca2+, activates a calcium/calmodulin (Ca2+/CaM)-dependent protein kinase which increases the phosphorylation of arrestin. We now have identified biochemically, a calcium/calmodulin-dependent protein phosphatase (Ca2+/CaM PP) in homogenates of the Limulus lateral and ventral eye, brain, and lateral optic nerve using as a substrate, a 32P-labeled peptide fragment of the regulatory subunit of cAMP-dependent protein kinase (RII). This protein phosphatase shares biochemical properties with calcineurin, a Ca2+/CaM-dependent protein phosphatase (type-2B). Its activity is enhanced by Ca2+, calmodulin and Mn2+; and is inhibited by mastoparan, a calmodulin antagonist, and a synthetic peptide corresponding to the autoinhibitory domain of mammalian calcineurin. Most importantly, light regulates the Ca2+/CaM PP activity in the lateral eye. While there is no difference in basal activity in long-term dark- or light-adapted preparations, Ca2+ enhances Ca2+/CaM PP activity only in long-term light-adapted eyes.
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PMID:Characterization of a calcium/calmodulin-dependent protein phosphatase in the Limulus nervous tissue and its light regulation in the lateral eye. 794 99

An arrestin homolog (Arr2, 49-kDa protein) of blowfly (Calliphora erythrocephala) retinae undergoes light-dependent reversible binding to the photoreceptor membrane. In order to characterize this arrestin homolog and to study its function in a well-defined experimental system, we developed a purification scheme which used microvillar photoreceptor membranes as an affinity binding matrix. Additional purification steps included ammonium sulfate precipitation, gel filtration and binding to heparin-agarose. The molecular mass of purified Arr2, as judged by SDS/PAGE, is in the range 45-49 kDa. The isoelectric point, as judged by gel isoelectric focussing, is 8.7. Arr2 is specific to the retina, where it is subject to phosphorylation at multiple sites. Binding of purified Arr2 to isolated photoreceptor membranes efficiently activates the light-induced phosphorylation of visual pigment. Since the assay system used is deficient in rhodopsin phosphatase activity, the arrestin-stimulated phosphate incorporation into rhodopsin results solely from the activation of a protein kinase. Phosphorylation experiments with highly purified membrane preparations indicate that rhodopsin kinase is tightly associated with the rhabdomeric membrane or the microvillar cytoskeleton. Rhodopsin kinase is released from the membrane or inactivated upon treatment with urea. It is concluded that this arrestin is a regulator protein that controls visual-pigment phosphorylation by affecting the interaction of metarhodopsin and rhodopsin (metarhodopsin) kinase.
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PMID:An arrestin homolog of blowfly photoreceptors stimulates visual-pigment phosphorylation by activating a membrane-associated protein kinase. 836 18

Receptor-specific or homologous desensitization of beta 2-adrenergic receptors is thought to be effected via phosphorylation of the receptor by the beta-adrenergic receptor kinase (beta ARK), followed by binding of beta-arrestin. We have generated stably transfected Chinese hamster ovary cell lines overexpressing either of the two regulatory proteins and also expressing low or high levels of beta 2-adrenergic receptors (approximately 80 and approximately 600 fmol/mg of membrane protein). In these cells, we studied the process of desensitization induced by the beta-adrenergic receptor agonist isoproterenol. In cells expressing high levels of beta 2-adrenergic receptors, desensitization to high concentrations of isoproterenol (previously shown to be mediated by both beta ARK and protein kinase A) amounted to approximately 50% in control cells, approximately 80% in beta ARK-overexpressing cells, and approximately 90% in beta-arrestin-overexpressing cells. In cells expressing low levels of beta 2-adrenergic receptors, these values were approximately 50, approximately 60, and approximately 60%, respectively. Desensitization to low concentrations of isoproterenol (previously shown to be essentially protein kinase A-mediated and not receptor-specific, i.e. heterologous) was not affected by overexpression of either beta ARK or beta-arrestin. These data suggest that in cells expressing high levels of beta 2-adrenergic receptors, beta-arrestin and beta ARK become limiting for homologous receptor desensitization. They provide further support for the involvement of these two proteins in the regulation of beta 2-adrenergic receptor function.
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PMID:Overexpression of beta-arrestin and beta-adrenergic receptor kinase augment desensitization of beta 2-adrenergic receptors. 838 21


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