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)

The gene for rhodopsin, the primary light sensor of the visual system, is specifically expressed in the rod photoreceptor cells of the retina. We show here that in the rat, opsin RNA first accumulates to detectable levels at postnatal day 2 (PN2) and that nascent transcripts can be detected at PN1; this is the time when peak numbers of photoreceptor cells are generated by the final division of their neuroepithelial precursors. Accumulated opsin RNA then increases to reach the adult level, 0.06% of total retinal RNA, at about PN10. The transcription rate of the opsin gene increases to a similar extent over the same time course between PN3 and adulthood, suggesting that transcriptional activation is responsible for the increase in opsin expression. We used the antibody RET-P1 to show that rhodopsin protein is also detectable at PN2 and that the number of cells expressing the protein increases with time in a central-to-peripheral gradient in the retina. This increase in the number of differentiating photoreceptors in the tissue appears to account for much of the increase in opsin gene transcription and RNA accumulation. In situ hybridization to opsin RNA shows that it is restricted to the photoreceptor layer from the time it can first be detected, at PN7. Later in development, when RET-P1 staining shifts to the photoreceptor outer segments, opsin RNA becomes localized to the inner segments, suggesting that the distributions of opsin protein and RNA are related.
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PMID:Opsin expression in the rat retina is developmentally regulated by transcriptional activation. 296 11

The structural components involved in transduction of extracellular signals as diverse as a photon of light impinging on the retina or a hormone molecule impinging on a cell have been highly conserved. These components include a recognition unit or receptor (for example, the beta-adrenergic receptor (beta AR) for catecholamines or the 'light receptor' rhodopsin), a guanine nucleotide regulatory or transducing protein, and an effector enzyme (for example, adenylate cyclase or cyclic GMP phosphodiesterase). Molecular cloning has revealed that the beta AR shares significant sequence and three-dimensional homology with rhodopsin. The function of the beta AR is diminished by exposure to stimulatory agonists, leading to desensitization. Similarly, 'light adaptation' involves decreased coupling of photoactivated rhodopsin to cGMP phosphodiesterase activation. Both forms of desensitization involve receptor phosphorylation. The latter is mediated by a unique protein kinase, rhodopsin kinase, which phosphorylates only the light-bleached form of rhodopsin. An analogous enzyme (termed beta AR kinase or beta ARK) phosphorylates only the agonist-occupied beta AR. We report here that beta ARK is also capable of phosphorylating rhodopsin in a totally light-dependent fashion. Moreover, rhodopsin kinase can phosphorylate the agonist-occupied beta AR. Thus the mechanisms which regulate the function of these disparate signalling systems also appear to be similar.
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PMID:Light-dependent phosphorylation of rhodopsin by beta-adrenergic receptor kinase. 301 40

Lectin and rhodopsin antibody binding sites were studied in developing and adult rat photoreceptors in order to compare changes in the total carbohydrate pool with the movement of a known glycoprotein rhodopsin. Electron microscope immunocytochemical techniques utilizing modified colloidal gold methods were used. At birth, all three lectins - Concanavalin A (ConA), Ricinus communis agglutinin II (RCA II) and wheat germ agglutinin (WGA) - showed heavy labelling of the photoreceptor surface scleral to the outer limiting membrane. At the same age, a monoclonal antibody against rhodopsin, RET-P1, revealed sparse labelling of only occasional immature photoreceptor surfaces. At postnatal day 4(P4), all three lectins showed variable binding to the inner segment and along the length of the newly forming connecting cilium. There was generally a region of more intense label at the base of the cilium. RET-P1 binding to P4 retina showed a discontinuous distribution, with heavily labelled inner segments being adjacent to unlabelled inner segments. This pattern indicates that the initial expression of rhodopsin is not a coordinate event but occurs in discrete cells, possibly related to the end of mitosis. RET-P1 binding at this age was reduced or absent from the proximal connecting cilium. AT P7, when the outer segments are beginning to develop, all the lectins and RET-P1 showed reduced binding to the inner segment plasma membrane and heavy labelling of the outer segment surface. In favourable sections, heavy labelling of the photoreceptor cell body plasma membrane by ConA and RCA II was also observed, terminating abruptly at the outer limiting membrane. The variation in ligand binding between different cellular compartments which are all formed from a continuous plasma membrane may indicate the presence of special barriers to diffusion of membrane components. This labelling pattern persisted into maturity. RET-P1 and lectin binding did not always correspond in developing retina, indicating that at least part of the observed lectin label must be due to other glycoproteins or glycolipids. Post-embedding thin section labelling of adult rat retina revealed a uniform binding pattern across the outer segment for ConA, WGA and RET-P1. However, RCA II exhibited labelling only along the basal edge of outer segments. Labelling of isolated, opened discs from bovine rod outer segments revealed binding to a single surface for ConA, WGA and RET-P1, but RCA II only labelled a small amount of membrane. Hence RCA II seems to recognize a determinant present only on the outer segment plasma membrane.
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PMID:Lectin and antibody labelling of developing rat photoreceptor cells: an electron microscope immunocytochemical study. 375 63

The subcellular localization of three photoreceptor antigens (RET-P1, rhodopsin and RET-P2) has been studied by electron microscopic immunocytochemistry of rat retinas. Localization was also examined by determining the amount of RET-P1 and RET-P2 antigen in various subcellular fractions. RET-P1 and RET-P2 antigens were further characterized by immunoblotting of crude retina membrane proteins which had been separated by one-dimensional gel electrophoresis. RET-P1 antigen has been detected with a monoclonal antibody that reacts with the perikarya, inner segments, and outer segments of adult rat photoreceptors by peroxidase immunolabelling of fixed tissue sections. Analysis at the electron microscopic level has shown that RET-P1 antigen is located on the external face of the inner and outer segment plasma membrane. A monoclonal antibody against purified bovine rhodopsin (RHO-C7) labels the outer segments of rat retinas by peroxidase immunocytochemistry. Ultrastructural antibody localization indicates that this particular determinant of rhodopsin is exposed on the external face of the plasma membrane of outer segments and may also be expressed on the surface of the inner segments. RET-P2 antibody labels only the outer segments of adult rat photoreceptors by peroxidase immunocytochemistry. The light microscopic labelling of RET-P2 antibody in the presence, but not in the absence, of detergent suggests that it is an intracellular antigen. The results of both ultrastructural labelling and biochemical fractionation are consistent with the localization of RET-P2 antigen on the internal face of the plasma membrane and/or the cytoplasmic face of the disc membranes. RET-P2 antigen was found to be a protein (or glycoprotein) of apparent molecular weight 38 000 +/- 3000.
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PMID:The subcellular localization of rat photoreceptor-specific antigens. 635 24

beta-arrestin is a cytosolic protein thought to be responsible for uncoupling agonist-activated beta 2-adrenergic receptors from their guanine-nucleotide-binding proteins (G-protein) subsequent to receptor phosphorylation by the beta-adrenergic receptor kinase (beta ARK). In order to investigate this interaction, we generated a recombinant baculovirus for the expression of beta-arrestin in Sf9 insect cells. Apparently homogeneous beta-arrestin preparations were obtained in a one-step purification on heparin-Sepharose. Purified beta-arrestin bound to rhodopsin in a phosphorylation-dependent plus light-dependent manner. Binding to beta 2-adrenergic receptors was investigated using purified receptors reconstituted into lipid vesicles. The accessibility of the reconstituted receptors was determined using the agonist isoproterenol for the ligand-binding site and an antibody binding to an attached myc tag for the C-terminus, the site of receptor phosphorylation. On the basis of these data, the binding of purified beta-arrestin to beta ARK-phosphorylated beta 2-adrenergic receptors was found to occur with a KD of 1.8 nM and with a maximum of 1 beta-arrestin/receptor. beta-arrestin also bound to receptors which had been completely dephosphorylated with acid phosphatase, but the affinity was approximately 30-fold lower. In contrast to regulation by phosphorylation, binding of agonists or antagonists to the receptors had negligible effects on beta-arrestin binding. Finally, beta-arrestin and beta ARK were shown to be capable of producing synergistic inhibition of beta 2-adrenergic-receptor-stimulated adenylyl cyclase activity of cell membranes. These data show that high-affinity stoichiometric binding of beta-arrestin to beta 2-adrenergic receptors occurs in a beta ARK-dependent manner and is sufficient to impair adenylyl cyclase stimulation by the receptors.
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PMID:Binding of purified recombinant beta-arrestin to guanine-nucleotide-binding-protein-coupled receptors. 755 95

The beta gamma subunits of heterotrimeric G proteins (G beta gamma) play a variety of roles in cellular signaling, one of which is membrane targeting of the beta-adrenergic receptor kinase (beta ARK). This is accomplished via a physical interaction of G beta gamma and a domain within the carboxyl terminus of beta ARK which overlaps with a pleckstrin homology (PH) domain. The PH domain of beta ARK not only binds G beta gamma but also interacts with phosphatidylinositol 4,5-bisphosphate (PIP2). Based on previous mapping of the G beta gamma binding region of beta ARK, and conserved residues within the PH domain, we have constructed a series of mutants in the carboxyl terminus of beta ARK in order to determine important residues involved in G beta gamma and PIP2 binding. To examine the effects of mutations on G beta gamma binding, we employed three different methodologies: direct G beta gamma binding to GST fusion proteins; the ability of GST fusion proteins to inhibit G beta gamma-mediated beta ARK translocation to rhodopsin-enriched rod outer segments; and the ability of mutant peptides expressed in cells to inhibit G beta gamma-mediated inositol phosphate accumulation. Direct PIP2 binding was also assessed on mutant GST fusion proteins. Ala residue insertion following Trp643 completely abolished the ability of beta ARK to bind G beta gamma, suggesting that a proper alpha-helical conformation is necessary for the G beta gamma.beta ARK interaction. In contrast, this insertional mutation had no effect on PIP2 binding. Both G beta gamma binding and PIP2 binding were abolished following Ala replacement of Trp643, suggesting that this conserved residue within the last subdomain of the PH domain is crucial for both interactions. Other mutations also produced differential effects on the physical interactions of the beta ARK carboxyl terminus with G beta gamma and PIP2. These results suggest that the last PH subdomain and its neighboring sequences within the carboxyl terminus of beta ARK, including Trp643, Leu647, and residues Lys663-Arg669, are critical for G beta gamma binding while Trp643 and residues Asp635-Glu639 are important for the PH domain to form the correct structure for binding to PIP2.
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PMID:Mutational analysis of the pleckstrin homology domain of the beta-adrenergic receptor kinase. Differential effects on G beta gamma and phosphatidylinositol 4,5-bisphosphate binding. 762 21

The aim of this study was to test the possible modification of beta-adrenergic receptor kinase (beta ARK) activity by second messengers and/or their downstream components. Using human mononuclear leukocytes (MNL), we found that calcium ionophores could elevate beta ARK activity by about 80% in a protein kinase C (PKC)-dependent manner. This was confirmed by the ability of the PKC activator phorbol 12-myristate 13-acetate (PMA) to produce a similar effect, suggesting a PKC-dependent modulation of beta ARK activity. In vitro experiments with purified proteins showed that PKC could directly phosphorylate beta ARK1 with an apparent Km for beta ARK1 of 6 nM. The ability of beta ARK1 to phosphorylate rhodopsin was 61% greater when it was phosphorylated by PKC. The level of phosphorylation of beta ARK1 immunoprecipitated from MNL and Sf9 cells overexpressing this kinase was enhanced by about 2-3-fold after PMA treatment. Functional significance of PKC-dependent increase in beta ARK activity ws demonstrated by beta-adrenergic receptor (beta AR) homologous desensitization experiments in MNL. beta AR desensitization, as induced by exposure to 10 microM isoproterenol (5 min at 37 degrees C), was increased from 42 +/- 10% in control to 68 +/- 8% in PMA-pretreated MNL. beta ARK inhibitor heparin (160 micrograms/ml) prevented the augmenting effect of PMA on beta AR desensitization. These results show that beta ARK activity can be increased through phosphorylation by PKC, thus indicating that beta ARK can be preconditioned to modulate the subsequent cellular responsiveness to receptor activation, providing the cell with a mechanism by which specific homologous desensitization can be regulated heterologously.
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PMID:Phosphorylation and activation of beta-adrenergic receptor kinase by protein kinase C. 762 97

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

Beta-adrenergic receptor kinase (beta ARK) is a serine-threonine kinase involved in the process of homologous desensitization of G-coupled receptors. beta ARK is a member of a multigene family, consisting of six known subtypes, also named G protein-coupled receptor kinases (GRK 1-6). In this study we investigated the expression of GRKs during the process of T cell activation, which is of fundamental importance in regulating immune responses. T cell activation was induced by exposing mononuclear leukocytes (MNL) to PHA and confirmed by tritiated thymidine incorporation measurement. A substantial increase of GRK activity (as measured by in vitro phosphorylation of rhodopsin) was found after 48 h (331 +/- 80% of controls) and 72 h (347 +/- 86% of controls) of exposure to PHA. A threefold increase of beta ARK1 immunoreactivity was found in MNL exposed to PHA for 72 h. Persistent activation of protein kinase C (PKC) by 10 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) was able to increase beta ARK activity to the same extent as PHA, suggesting a PKC-mediated mechanism. The kinetic of beta-adrenergic-stimulated cAMP production was substantially modified in TPA and PHA-activated cells, indicating that the increased GRK activity resulted in an increased beta-adrenergic homologous desensitization. A three- to fourfold increase in GRK activity was also observed in a population of T cell blasts (> 97% CD3+) exposed to PHA for 48-72 h. A significant increase in beta ARK1 and beta ARK2 mRNA expression was observed 48 h after mitogen stimulation, while mRNA expression of GRK5 and GRK6 was not changed. In conclusion our data show that the expression of GRK subtypes is actively and selectively modulated according to the functional state of T lymphocytes.
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PMID:Regulation of G protein-coupled receptor kinase subtypes in activated T lymphocytes. Selective increase of beta-adrenergic receptor kinase 1 and 2. 781 17

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


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