Abstract
We reported previously that ethanol treatment regulates D1 receptor phosphorylation and signaling in a protein kinase C (PKC) δ- and PKCγ-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 PKCγ and PKCδ. Both RanBP9 and RanBP10 were found to specifically coimmunoprecipitate with both PKCγ and PKCδ; however, this association did not seem to mediate the ethanol regulation of the PKCs. It is noteworthy that the D1 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 D1 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 PKCδ or PKCγ, both RanBP proteins regulated D1 receptor phosphorylation, signaling, and, in the case of RanBP9, expression. Specifically, overexpression of either RanBP9 or RanBP10 enhanced basal D1 receptor phosphorylation, which was associated with attenuation of D1 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 D1 receptor signaling by PKCδ and PKCγ.
Footnotes
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material. -
This research was supported by the National Institutes of Health National Heart, Lung, and Blood Institute [Grants R37-HL023081, P01-HL074940, P01-HL068686, R01-HL092196]; the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [R01-DK039308]; and Intramural Research funds from the National Institutes of Health National Institute of Neurological Disorders and Stroke and the National Institutes of Health National Institute of Mental Health.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.110.063727.
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ABBREVIATIONS:
- PKC
- protein kinase C
- PKA
- protein kinase A
- GPCR
- G protein-coupled receptor
- HEK
- human embryonic kidney
- DA
- dopamine
- GFP
- green fluorescent protein
- PAGE
- polyacrylamide gel electrophoresis
- DMEM
- Dulbecco's modified essential medium
- FBS
- fetal bovine serum
- PVDF
- polyvinylidene difluoride
- EtOH
- ethanol
- mGluR
- metabotropic glutamate receptor
- MOR
- μ-opioid receptor
- GRK
- G protein-coupled receptor kinase
- PBS
- phosphate-buffered saline
- HA
- hemagglutinin
- MOPS
- 3-(N-morpholino)propanesulfonic acid
- DAG
- diacylglycerol
- PS
- phosphatidyl serine
- coIP
- coimmunoprecipitation
- D1R
- dopamine 1 receptor
- EGFP
- enhanced green fluorescent protein
- Hsp-70
- 70-kDa heat shock protein
- ERK
- extracellular signal-regulated kinase
- PICK1
- protein interacting with C kinase 1
- RACK
- receptor for activated C kinase
- SCH23390
- R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine
- Gö6976
- 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole
- Gö6983
- 3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione
- Ro 20-1724
- 4-[3-butoxy-4-methoxyphenyl)-methyl]-2-imidazolidinone.
- Received January 22, 2010.
- Accepted April 15, 2010.
- U.S. Government work not protected by U.S. copyright
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