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of special interest. The Kaposi's sarcoma-associated herpesvirus (KSVH)-GPR represents a novel constitutively active GPR that promotes cell transformation and tumorigenicity through activation of JNK/SAPK and p38MAPK. In NIH-3T3 cells, KSVH-GPR-induced cell proliferation was specifically inhibited by overexpression of GRK5, suggesting a potential therapeutic avenue to be explored for such GPR-dependent tumors.
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Bais C, Bianca S, Coso O, Arvanitakis L, Raaka GE, Gutkind JS, Asch AJ, Cesarman E, Gerhengorn MC, Mesri EA. G-protein-coupled receptor of Kaposi's sarcoma-associated herpesvirus is a viral oncogene and angiogenesis activator. of special interest Nature. 391:1998;86-89 The Kaposi's sarcoma-associated herpesvirus (KSVH)-GPR represents a novel constitutively active GPR that promotes cell transformation and tumorigenicity through activation of JNK/SAPK and p38MAPK. In NIH-3T3 cells, KSVH-GPR-induced cell proliferation was specifically inhibited by overexpression of GRK5, suggesting a potential therapeutic avenue to be explored for such GPR-dependent tumors.
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of special interest. The GRK2 gene was disrupted by homologous recombination in mice leading to embryonic death at around day 15 attributed to severe hypoplasia of the ventricular myocardium. This phenotype is interestingly similar to that of 'thin myocardial syndrome' which is associated with disruption of several transcription factors (RXRα, N-Myc, TEF-1, WT-1). This study demonstrates an important role for GRK2 in development.
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Jaber M, Koch WJ, Rockman H, Smith B, Bond RA, Sulik KK, Ross J Jr, Lefkowitz RJ, Caron MG, Giros B. Essential role of β-adrenergic receptor kinase in cardiac development and function. of special interest Proc Natl Acad Sci USA. 93:1996;12974-12979 The GRK2 gene was disrupted by homologous recombination in mice leading to embryonic death at around day 15 attributed to severe hypoplasia of the ventricular myocardium. This phenotype is interestingly similar to that of 'thin myocardial syndrome' which is associated with disruption of several transcription factors (RXRα, N-Myc, TEF-1, WT-1). This study demonstrates an important role for GRK2 in development.
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Jaber, M.1
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The Drosophila G protein-coupled receptor kinase homologue Gprk2 is required for egg morphogenesis
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of special interest. A mutation in Drosophila Gprk2 (a GRK4 subfamily homolog) was identified and shown to disrupt expression of Gprk2 in the central nervous system and ovaries of females, rendering them sterile. However, rare eggs that did become fertilized displayed gross defects in embryogenesis, primarily associated with anterior chorion formation, suggesting a role for GRKs in regulation of growth and development.
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of special interest Schneider LE, Spradling AC. The Drosophila G protein-coupled receptor kinase homologue Gprk2 is required for egg morphogenesis. Development. 124:1997;2591-2602 A mutation in Drosophila Gprk2 (a GRK4 subfamily homolog) was identified and shown to disrupt expression of Gprk2 in the central nervous system and ovaries of females, rendering them sterile. However, rare eggs that did become fertilized displayed gross defects in embryogenesis, primarily associated with anterior chorion formation, suggesting a role for GRKs in regulation of growth and development.
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Schneider, L.E.1
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G protein-coupled receptor kinase 3 (GRK3) gene disruption leads to loss of odorant receptor desensitization
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of special interest. Targeted gene disruption was performed to produce GRK-3 deficient mice which were normal in embryonic and postnatal development. However, cilia preparations from the olfactory epithelium, which normally express an abundance of GRK3 but not GRK2, were obtained from the GRK3-deficient mice and shown to lack the fast agonist-induced desensitization seen after odorant stimulation in cilia from wild-type mice.
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Peppel K, Boekhoff I, McDonald P, Breer H, Caron MG, Lefkowitz RJ. G protein-coupled receptor kinase 3 (GRK3) gene disruption leads to loss of odorant receptor desensitization. of special interest J Biol Chem. 272:1997;25425-25428 Targeted gene disruption was performed to produce GRK-3 deficient mice which were normal in embryonic and postnatal development. However, cilia preparations from the olfactory epithelium, which normally express an abundance of GRK3 but not GRK2, were obtained from the GRK3-deficient mice and shown to lack the fast agonist-induced desensitization seen after odorant stimulation in cilia from wild-type mice.
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of special interest. Rod cells were isolated from arrestin-deficient mice, generated by targeted gene disruption, allowing measurement of single-cell photoresponses. These experiments revealed that the initial kinetics of recovery, probably governed by rhodopsin kinase, were normal while the final phase of recovery was significantly prolonged in the absence of functional arrestin.
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of special interest. GRK2-mediated binding and phosphorylation of purified GST - m2AChR and - m3AChR third intracellular loop fusion proteins was examined in the absence and presence of G-protein βγ subunits. These experiments demonstrate βγ-dependent enhancement of both binding and phosphorylation suggesting that βγ may directly target GRK2 to receptor substrates via formation of a GPR/GRK2/βγ ternary complex.
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Wu G, Benovic JL, Hildebrandt JD, Lanier SM. Intracellular motifs of G-protein coupled receptors present docking sites for G-protein βγ subunits. of special interest J Biol Chem. 273:1998;7197-7200 GRK2-mediated binding and phosphorylation of purified GST - m2AChR and - m3AChR third intracellular loop fusion proteins was examined in the absence and presence of G-protein βγ subunits. These experiments demonstrate βγ-dependent enhancement of both binding and phosphorylation suggesting that βγ may directly target GRK2 to receptor substrates via formation of a GPR/GRK2/βγ ternary complex.
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2ARs and m2AChRs reveals that these GPRs bind agonists (but not antagonists) with higher affinity when complexed with an arrestin. These studies also demonstrate selectivity among various arrestins and receptors with respect to this phenomenon, which may reflect receptor- and arrestin-dependent differences in the propensity of arrestins to undergo transition into the active receptor-binding state.
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2ARs and m2AChRs reveals that these GPRs bind agonists (but not antagonists) with higher affinity when complexed with an arrestin. These studies also demonstrate selectivity among various arrestins and receptors with respect to this phenomenon, which may reflect receptor- and arrestin-dependent differences in the propensity of arrestins to undergo transition into the active receptor-binding state.
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A β-arrestin/green fluorescent protein biosensor for detecting G protein-coupled receptor activation
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of special interest. To investigate cellular GPR - arrestin interaction, an arrestin-3-GFP construct, GRK2, and various GPRs were co-transfected into HEK-293 and COS-7 cells. This system provides 'real-time' visualization of the dynamic recruitment of cytosolic arrestin-3 to the plasma membrane upon receptor activation and illuminates the critical role of GRKs in supporting this process. Importantly, arrestin-3 mobilization was shown to be elicited by activation of 15 distinct GPRs, suggesting the 'universality' of this regulatory process.
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Barak LS, Ferguson SSG, Zhang J, Caron MG. A β-arrestin/green fluorescent protein biosensor for detecting G protein-coupled receptor activation. of special interest J Biol Chem. 272:1997;27497-27500 To investigate cellular GPR - arrestin interaction, an arrestin-3-GFP construct, GRK2, and various GPRs were co-transfected into HEK-293 and COS-7 cells. This system provides 'real-time' visualization of the dynamic recruitment of cytosolic arrestin-3 to the plasma membrane upon receptor activation and illuminates the critical role of GRKs in supporting this process. Importantly, arrestin-3 mobilization was shown to be elicited by activation of 15 distinct GPRs, suggesting the 'universality' of this regulatory process.
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Barak, L.S.1
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Krupnick JG, Gurevich VV, Benovic JL. Mechanism of quenching phototransduction: Binding competition between arrestin and transducin for phosphorhodopsin. J Biol Chem. 272:1997;18125-18131.
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Mechanism of phosphorylation-recognition by visual arrestin and the transition of arrestin into a high affinity binding state
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of special interest. The critical phosphorylation-recognition residue Arg175, as well as adjacent conserved hydrophobic residues in arrestin were extensively mutagenized and then assessed for their ability to bind phosphorylated, light-activated rhodopsin as well as phosphorylation-site-deficient rhodopsin. These studies reveal two relatively independent changes that occur in arrestin on receptor binding: increased binding affinity of the phosphorylation-recognition region for the phosphorylated carboxyl-termianl tail of rhodopsin; and mobilization of additional receptor-binding sites. These experiments also reveal that charge inversion at position 175 generates a 'constitutively active' arrestin that demonstrates phosphorylation-independent receptor binding.
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Gurevich VV, Benovic JL. Mechanism of phosphorylation-recognition by visual arrestin and the transition of arrestin into a high affinity binding state. of special interest Mol Pharmacol. 51:1997;161-169 The critical phosphorylation-recognition residue Arg175, as well as adjacent conserved hydrophobic residues in arrestin were extensively mutagenized and then assessed for their ability to bind phosphorylated, light-activated rhodopsin as well as phosphorylation-site-deficient rhodopsin. These studies reveal two relatively independent changes that occur in arrestin on receptor binding: increased binding affinity of the phosphorylation-recognition region for the phosphorylated carboxyl-termianl tail of rhodopsin; and mobilization of additional receptor-binding sites. These experiments also reveal that charge inversion at position 175 generates a 'constitutively active' arrestin that demonstrates phosphorylation-independent receptor binding.
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i, with or without β-agonist pretreatment. This revealed constitutive phosphorylation of Ser412 on β-arrestin, as well as agonist-dependent and membrane-localized β-arrestin dephosphorylation. It was demonstrated that the phosphorylated arrestin, as well as a β-arrestin-S412D mutant were competent for receptor binding and desensitization but unable to bind clathrin and promote receptor sequestration, suggesting a possible role for phosphorylation in modulating arrestin activity.
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2ARs whereas these processes were profoundly and modestly inhibited by overexpression of dynamin-K44A and a β-arrestin(319-418) respectively, demonstrating a direct link between sequestration and downregulation mechanisms in these cells. Interestingly, similar experiments in COS-1 and HeLa cells revealed qualitatively similar but quantitatively distinct relationships between sequestration and downregulation, underscoring the importance of cellular components in these processes.
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of special interest. GRK activity and expression was assessed by rhodopsin phosphorylation and immunoblotting, respectively, in lymphocytes of both hypertensive and normotensive subjects. These studies reveal increased GRK activity and a selective increase in GRK2 expression that may underlie the reduce β-adrenergic responsiveness characteristic of the hypertensive state.
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of special interest. Rabbits with tachycardia-induced CHF were shown to exhibit a physiological reduction in myocardial function characteristic of CHF, as well as decreased βAR signalling (~twofold), increased GRK2 expression (~twofold) and increased GRK activity (~threefold). Adenovirus-mediated expression of a GRK2 minigene inhibitor in cultured left ventricular myocytes from failing hearts restored βAR-mediated cAMP production to levels equal to or greater than those of control myocytes, demonstrating that the reduced βAR signalling is attributable to increased GRK2 activity.
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Akhter SA, Skaer CA, Kypson AP, McDonald PH, Peppel KC, Glower DD, Lefkowitz RJ, Koch RJ. Restoration of β-adrenergic signaling in falling cardiac ventricular myocytes via adenoviral-mediated gene transfer. of special interest Proc Natl Acad Sci USA. 94:1997;12100-12105 Rabbits with tachycardia-induced CHF were shown to exhibit a physiological reduction in myocardial function characteristic of CHF, as well as decreased βAR signalling (~twofold), increased GRK2 expression (~twofold) and increased GRK activity (~threefold). Adenovirus-mediated expression of a GRK2 minigene inhibitor in cultured left ventricular myocytes from failing hearts restored βAR-mediated cAMP production to levels equal to or greater than those of control myocytes, demonstrating that the reduced βAR signalling is attributable to increased GRK2 activity.
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75
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of special interest. Electroretinography and psychophysics were used to measure in vivo photoresponses in an Oguchi patient shown to be homozygous for a rhodopsin kinase deletion thought to generate a null phenotype. These data demonstrate rod recovery phase abnormalities that interestingly slowed both rod and cone deactivation kinetics.
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Cidecyan AV, Zhao X, Neilsen L, Khani SC, Jacobson SG, Palczewski K. Null mutation in the rhodopsin kinase gene slows recovery kinetics of rod and cone phototransduction in man. of special interest Proc Natl Acad Sci USA. 95:1998;328-333 Electroretinography and psychophysics were used to measure in vivo photoresponses in an Oguchi patient shown to be homozygous for a rhodopsin kinase deletion thought to generate a null phenotype. These data demonstrate rod recovery phase abnormalities that interestingly slowed both rod and cone deactivation kinetics.
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Cidecyan, A.V.1
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