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:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

During short term agonist exposure, the alpha 2A-adrenergic receptor (alpha 2AAR) undergoes rapid functional desensitization caused by phosphorylation of the receptor by the beta-adrenergic receptor kinase (beta ARK). This signal quenching is similar in nature to that found with a number of G-protein coupled receptors in which agonist-promoted desensitization is due to beta ARK phosphorylation; like these other receptors, the precise molecular determinants of the receptor required for beta ARK phosphorylation are not known. To delineate such a motif in the human alpha 2AAR (alpha 2C10), we constructed six mutated receptors consisting of deletions or substitutions of Ser-296-299 in the EESSSS sequence of the third intracellular loop of the receptor. These were expressed in Chinese hamster ovary and COS-7 cells, and agonist-promoted desensitization and receptor phosphorylation were assessed. Deletion of the EESSSS sequence and substitution of alanine for all four serines resulted in a total loss of phosphorylation and desensitization. Mutant receptors that retained two of the original serines (AASS and SSAA) underwent agonist-promoted phosphorylation of 55 +/- 7% and 57 +/- 8% of the phosphorylation found for wild type alpha 2C10. Additional substitution mutants (SSSA and SAAA) underwent 77 +/- 1% and 27 +/- 4% of wild type phosphorylation, respectively. Thus, substitution of alanine for each additional serine decreased overall phosphorylation as compared with wild type alpha 2C10 by approximately 25%, which is consistent with all 4 serines being phosphorylated. Mutated receptors that only partially phosphorylated (as compared with wild type) failed to undergo agonist-promoted desensitization. Thus, beta ARK-mediated phosphorylation of alpha 2C10 occurs at Ser-296-299 in the third intracellular loop, and this represents the critical step in rapid agonist-promoted desensitization. A number of other G-protein coupled receptors that undergo desensitization have a sequence motif similar to that which we have found for beta ARK-mediated phosphorylation of alpha 2C10, suggesting that these receptors may also be substrates for beta ARK.
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PMID:Four consecutive serines in the third intracellular loop are the sites for beta-adrenergic receptor kinase-mediated phosphorylation and desensitization of the alpha 2A-adrenergic receptor. 787 39

A diverse array of molecules involved in signal transduction have recently been recognised as containing a new homology domain, the pleckstrin homology (PH) domain. These include kinases (both serine/threonine and tyrosine specific), all currently known mammalian phospholipase Cs, GTPases, GTPase-activating proteins, GTPase-exchange factors, "adapter" proteins, cytoskeletal proteins, and kinase substrates. This has sparked a new surge of research into elucidating its structure and function. The NMR solution structure of the PH domains of beta-spectrin and pleckstrin (the N-terminal domain) both display a core consisting of seven anti-parallel beta-sheet strands. The carboxy terminus is folded into a long alpha-helix. The molecule is electrostatically polarised and contains a pocket which may be involved in the binding of a ligand. The PH domains overall topological relatedness to the retinoid binding protein family of molecules would suggest a lipid ligand could bind to this pocket. The prime function of the PH domain still remains to be elucidated. However, it has been shown to be important in signal transduction, most probably by mediating protein-protein interactions. An extended PH domain of the beta-adrenergic receptor kinase (beta ARK), as well as that of several other molecules, can bind to beta gamma subunits of the heterotrimeric G-proteins. The possibility that the PH domain, which is found in so many signalling molecules, being generally involved in beta gamma binding is provocative of implicating these proteins in G-protein signal transduction.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Pleckstrin homology (PH) domains in signal transduction. 789 Aug 2

Two types of proteins play a major role in determining homologous desensitization of G-coupled receptors: beta-adrenergic receptor kinase (beta ARK), which phosphorylates the agonist-occupied receptor, and its functional cofactor, beta-arrestin. beta ARK is a member of a multigene family, consisting of six known subtypes, which have also been named G-protein-coupled receptor kinases (GRK 1 to 6) due to the apparently unique functional association of such kinases with this receptor family. The gene for beta ARK1 has been localized to human chromosome 11q13. The four members of the arrestin/beta-arrestin gene family identified so far are arrestin, X-arrestin, beta-arrestin 1, and beta-arrestin 2. Here we report the chromosome mapping of the human gene for beta-arrestin 1 (ARRB1) to chromosome 11q13 by fluorescence in situ hybridization (FISH). Two-color FISH confirmed that the two genes coding for the functionally related proteins beta ARK1 and beta-arrestin 1 both map to 11q13.
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PMID:Assignment of the beta-arrestin 1 gene (ARRB1) to human chromosome 11q13. 789 72

Based on the established role of beta-adrenergic receptor kinase (beta ARK) and beta-arrestin in the desensitization of several G protein-coupled receptors, we investigated the effect of chronic morphine administration on beta ARK and beta-arrestin levels in selected brain areas. Levels of beta ARK were measured by blot immunolabeling analysis using antibodies specific for two known forms of beta ARK, i.e., beta ARK1 and beta ARK2. It was found that chronic morphine treatment produced an approximately 35% increase in levels of beta ARK1 immunoreactivity in the locus coeruleus, but not in several other brain regions studied. In contrast, chronic morphine treatment failed to alter levels of beta ARK2 immunoreactivity in any of the brain regions studied. Levels of beta-arrestin immunoreactivity, measured using an antiserum that recognizes two major forms of this protein in brain, were also found to increase (by approximately 20%) in the locus coeruleus. It is proposed that chronic morphine regulation of beta ARK1 and beta-arrestin levels may contribute to opioid-receptor tolerance that is known to occur in this brain region.
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PMID:Chronic morphine administration increases beta-adrenergic receptor kinase (beta ARK) levels in the rat locus coeruleus. 793 57

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

G protein-coupled receptor kinases (GRKs), such as rhodopsin kinase and beta-adrenergic receptor kinase (beta ARK), are involved in mediating agonist-specific phosphorylation and desensitization of G protein-coupled receptors. GRK6 is the most recently identified member of the GRK family and displays higher homology with GRK5 (70.1% amino acid identity) and IT11 (68.5%) compared to beta ARK (37.4%) and rhodopsin kinase (47.1%). To further characterize GRK6, it has been overexpressed in Sf9 cells and purified to homogeneity by sequential chromatography on SP-Sepharose and heparin-Sepharose columns. GRK6 shares a number of in vitro characteristics with GRK5, including potent inhibition by heparin and dextran sulfate (IC50 values of approximately 15 and approximately 7 nM, respectively), hyperstimulation by polycations, and preference for phosphorylation of non-acidic peptides. Rhodopsin and the beta 2-adrenergic and m2 muscarinic cholinergic receptors serve as stimulus-dependent substrates for GRK6, but with stoichiometries significantly lower than achieved by GRK5 and beta ARK. Additionally, GRK6 does not undergo significant autophosphorylation even though it contains residues identical to those that are autophosphorylated in GRK5 and rhodopsin kinase. These data extend our knowledge of a growing family of receptor-specific kinases and suggest that GRK6 has a substrate specificity distinct from beta ARK, rhodopsin kinase, and GRK5.
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PMID:Expression, purification, and characterization of the G protein-coupled receptor kinase GRK6. 807 21

The beta-adrenergic receptor kinase (beta ARK) specifically phosphorylates the activated form of multiple receptors such as the beta 2-adrenergic receptor (beta 2 AR) and rhodopsin. beta ARK also phosphorylates synthetic peptides, albeit with an approximately 10(4)-10(7)-fold lower Vmax/Km ratio as compared to receptors, with a clear preference for peptides containing acidic residues on the aminoterminal side of a serine or threonine. To further characterize the mechanism of substrate phosphorylation by beta ARK, we designed a series of analogue peptides containing a single amino acid change (serine, glutamic acid, or phosphoserine) situated 2 or 4 residues amino-terminal to the target serine. While beta ARK weakly phosphorylated peptides lacking an acidic residue, peptides containing either a single phosphoserine or glutamic acid were substantially better substrates with a 3.5- to 8-fold increase in Vmax. Additional studies demonstrated that the interaction of beta ARK with an activated receptor (beta 2AR* or Rho*) also significantly enhanced peptide phosphorylation. Both Rho* and a truncated rhodopsin lacking its carboxyl-terminal phosphorylation sites activated peptide phosphorylation to a similar extent with EC50 values for activation of 0.65 and 1.34 microM, respectively. In contrast, the agonist-occupied beta 2AR activated peptide phosphorylation by beta ARK with a substantially higher affinity (EC50 of 0.012 microM) compared to Rho*. The Vmax/Km ratio for beta ARK phosphorylation of a poor peptide substrate such as RRRASAAASAA was increased up to approximately 200-fold by the activated receptor while the phosphorylation of a good peptide substrate (RRREEEEESAAA) was increased only up to approximately 8-fold. Our results suggest that acidic residues (glutamic acid or phosphoserine) localized on the amino-terminal side of target serines are important but not essential determinants in directing peptide phosphorylation. The substrate specificity of beta ARK appears to rely more strongly on the overall topological structure of the activated receptor which promotes the specific binding and activation of beta ARK.
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PMID:Beta-adrenergic receptor kinase. Agonist-dependent receptor binding promotes kinase activation. 809 17

The beta gamma subunits (G beta gamma) of heterotrimeric G proteins modulate the activity of several signal-transducing effector molecules including G protein-coupled receptor kinases. G beta gamma binds to the carboxyl terminus of the beta-adrenergic receptor kinase (beta ARK) and regulates its activity. To investigate the effect of such a G beta gamma-binding domain on heterologous G beta gamma interactions, various receptors that can stimulate phospholipase C and/or type II adenylate cyclase were coexpressed in COS-7 cells with the carboxyl terminus of beta ARK1. Phosphoinositol hydrolysis in response to activation of receptors that stimulate phospholipase C via Gi beta gamma (alpha 2-adrenergic and M2-muscarinic cholinergic receptors) was markedly inhibited by the coexpressed beta ARK1 polypeptide, whereas that mediated by Gq alpha subunits (alpha 1-adrenergic and M1-muscarinic cholinergic receptors) was unaffected. Increased cellular cAMP levels due to stimulation of receptors and coexpressed adenylate cyclase II displayed marked inhibition in the presence of the beta ARK1 polypeptide. Moreover, inhibition of adenylate cyclase produced by alpha 2-adrenergic receptor stimulation (a Gi alpha-mediated process) was unaffected, indicating that the beta ARK1 polypeptide provides a useful tool for distinguishing between G alpha and G beta gamma pathways.
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PMID:Cellular expression of the carboxyl terminus of a G protein-coupled receptor kinase attenuates G beta gamma-mediated signaling. 811 63

Ligand-induced activation of many receptors leads to dissociation of the alpha- and beta gamma-subunit complexes of heterotrimeric G proteins, both of which regulate a variety of effector molecules involved in cellular signaling processes. In one case, a cytosolic enzyme, the beta-adrenergic receptor kinase (beta ARK) binds to the dissociated, prenylated, membrane-anchored beta gamma-subunits of heterotrimeric G proteins (G beta gamma) and is thereby targeted to its membrane-bound receptor substrate. Quite recently, numerous proteins involved in cellular signal transduction have been shown to contain sequences homologous with a "domain" originally identified in the protein "pleckstrin" (pleckstrin homology domain; PH domain) and subsequently found in the G beta gamma interaction region of the beta ARK sequence. Here we demonstrate that glutathione S-transferase-fusion proteins, containing sequences encompassing the PH domain of nine proteins from this group, bind G beta gamma to varying extents. Binding of G beta gamma to these fusion proteins was documented either by a direct binding assay or by ability to block G beta gamma-mediated membrane translocation of beta ARK1. G beta gamma binding to these fusion proteins was inhibited by the alpha subunit of Go (Go alpha), indicating that the binding of G beta gamma to G alpha and the PH domain-containing fusion proteins is mutually exclusive. Studies with a series of truncated PH domains derived from the Ras-guanine-nucleotide-releasing factor indicate that the G beta gamma binding domain includes only the C-terminal portion of the PH domain and sequences just distal to this. Protein-protein interactions between G beta gamma and PH domain-containing proteins may play a significant role in cellular signaling analogous to that previously demonstrated for Src homology 2 and 3 domains.
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PMID:Binding of G protein beta gamma-subunits to pleckstrin homology domains. 814 1


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