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Identification of RanBP 9/10 as interacting partners for protein kinase C (PKC) gamma/delta and the D1 dopamine receptor: regulation of PKC-mediated receptor phosphorylation.

ABSTRACT: We reported previously that ethanol treatment regulates D(1) receptor phosphorylation and signaling in a protein kinase C (PKC) delta- and PKCgamma-dependent fashion by a mechanism that may involve PKC isozyme-specific interacting proteins. Using a PKC isozyme-specific coimmunoprecipitation approach coupled to mass spectrometry, we report the identification of RanBP9 and RanBP10 as novel interacting proteins for both PKCgamma and PKCdelta. Both RanBP9 and RanBP10 were found to specifically coimmunoprecipitate with both PKCgamma and PKCdelta; however, this association did not seem to mediate the ethanol regulation of the PKCs. It is noteworthy that the D(1) receptor was also found to specifically coimmunoprecipitate with RanBP9/10 from human embryonic kidney (HEK) 293T cells and with endogenous RanBP9 from rat kidney. RanBP9 and RanBP10 were also found to colocalize at the cellular level with the D(1) receptor in both kidney and brain tissue. Although overexpression of RanBP9 or RanBP10 in HEK293T cells did not seem to alter the kinase activities of either PKCdelta or PKCgamma, both RanBP proteins regulated D(1) receptor phosphorylation, signaling, and, in the case of RanBP9, expression. Specifically, overexpression of either RanBP9 or RanBP10 enhanced basal D(1) receptor phosphorylation, which was associated with attenuation of D(1) receptor-stimulated cAMP accumulation. Moreover, treatment of cells with select PKC inhibitors blocked the RanBP9/10-dependent increase in basal receptor phosphorylation, suggesting that phosphorylation of the receptor by PKC is regulated by RanBP9/10. These data support the idea that RanBP9 and RanBP10 may function as signaling integrators and dictate the efficient regulation of D(1) receptor signaling by PKCdelta and PKCgamma.

SUBMITTER: Rex EB

PROVIDER: S-EPMC2912060 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Identification of RanBP 9/10 as interacting partners for protein kinase C (PKC) gamma/delta and the D1 dopamine receptor: regulation of PKC-mediated receptor phosphorylation.

Rex Elizabeth B EB Rankin Michele L ML Yang Yu Y Lu Quansheng Q Gerfen Charles R CR Jose Pedro A PA Sibley David R DR

Molecular pharmacology 20100415 1

We reported previously that ethanol treatment regulates D(1) receptor phosphorylation and signaling in a protein kinase C (PKC) delta- and PKCgamma-dependent fashion by a mechanism that may involve PKC isozyme-specific interacting proteins. Using a PKC isozyme-specific coimmunoprecipitation approach coupled to mass spectrometry, we report the identification of RanBP9 and RanBP10 as novel interacting proteins for both PKCgamma and PKCdelta. Both RanBP9 and RanBP10 were found to specifically coimm ...[more]

PMID: 20395553

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Project description:Homologous desensitization of D(1) dopamine receptors is thought to occur through their phosphorylation leading to arrestin association which interdicts G protein coupling. In order to identify the relevant domains of receptor phosphorylation, and to determine how this leads to arrestin association, we created a series of mutated D(1) receptor constructs. In one mutant, all of the serine/threonine residues within the 3rd cytoplasmic domain were altered (3rdTOT). A second construct was created in which only three of these serines (serines 256, 258, and 259) were mutated (3rd234). We also created four truncation mutants of the carboxyl terminus (T347, T369, T394, and T404). All of these constructs were comparable with the wild-type receptor with respect to expression and adenylyl cyclase activation. In contrast, both of the 3rd loop mutants exhibited attenuated agonist-induced receptor phosphorylation that was correlated with an impaired desensitization response. Sequential truncation of the carboxyl terminus of the receptor resulted in a sequential loss of agonist-induced phosphorylation. No phosphorylation was observed with the most severely truncated T347 mutant. Surprisingly, all of the truncated receptors exhibited normal desensitization. The ability of the receptor constructs to promote arrestin association was evaluated using arrestin-green fluorescent protein translocation assays and confocal fluorescence microscopy. The 3rd234 mutant receptor was impaired in its ability to induce arrrestin translocation, whereas the T347 mutant was comparable with wild type. Our data suggest a model in which arrestin directly associates with the activated 3rd cytoplasmic domain in an agonist-dependent fashion; however, under basal conditions, this is sterically prevented by the carboxyl terminus of the receptor. Receptor activation promotes the sequential phosphorylation of residues, first within the carboxyl terminus and then the 3rd cytoplasmic loop, thereby dissociating these domains and allowing arrestin to bind to the activated 3rd loop. Thus, the role of receptor phosphorylation is to allow access of arrestin to its receptor binding domain rather than to create an arrestin binding site per se.

Dataset Information

Identification of RanBP 9/10 as interacting partners for protein kinase C (PKC) gamma/delta and the D1 dopamine receptor: regulation of PKC-mediated receptor phosphorylation.

Publications

Identification of RanBP 9/10 as interacting partners for protein kinase C (PKC) gamma/delta and the D1 dopamine receptor: regulation of PKC-mediated receptor phosphorylation.

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