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

Mol Pharmacol. 2010 Jul;78(1):69-80. doi: 10.1124/mol.110.063727. Epub 2010 Apr 15.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural

MeSH terms

  • Adaptor Proteins, Signal Transducing / metabolism*
  • Animals
  • Cell Line
  • Cyclic AMP / metabolism
  • Cytoskeletal Proteins / metabolism*
  • Guanine Nucleotide Exchange Factors / metabolism*
  • Humans
  • Microscopy, Confocal
  • Microtubule-Associated Proteins / metabolism*
  • Neurons / metabolism
  • Nuclear Proteins / metabolism*
  • Phosphorylation
  • Protein Binding
  • Protein Kinase C / metabolism*
  • Protein Kinase C-delta / metabolism*
  • Rats
  • Receptors, Dopamine D1 / metabolism*

Substances

  • Adaptor Proteins, Signal Transducing
  • Cytoskeletal Proteins
  • Guanine Nucleotide Exchange Factors
  • Microtubule-Associated Proteins
  • Nuclear Proteins
  • RANBP10 protein, human
  • Ran binding protein 9
  • Receptors, Dopamine D1
  • Cyclic AMP
  • protein kinase C gamma
  • Protein Kinase C
  • Protein Kinase C-delta