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J. Biol. Chem., Vol. 283, Issue 51, 35908-35917, December 19, 2008

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GAP1(IP₄BP)/RASA3 Mediates G_i-induced Inhibition of Mitogen-activated Protein Kinase^*

From the Ottawa Health Research Institute (Neuroscience), Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa K1H 8M5, Canada

The dopamine D2S receptor (short isoform) couples to inhibitory G_i/o proteins to inhibit thyrotropin-releasing hormone (TRH)-stimulated p42/p44 mitogen-activated protein kinase (ERK1/2) phosphorylation in GH4ZR7 rat pituitary cells, consistent with its actions to inhibit prolactin gene transcription and cell proliferation. However, the underlying mechanism is unclear. To identify novel G_i effectors, yeast two-hybrid screening of a GH4ZR7 cDNA library was done using constitutively active G_i3-Q204L, and multiple clones of the RasGAP cDNA GAP1^IP4BP/RASA3 were identified. In yeast mating assay, RASA3 preferentially interacted with activated forms of G_i/o/z proteins, but not with G_s. A direct interaction was indicated by in vitro pull-down assay, in which S-His-RASA3 preferentially bound guanosine 5'-O-(-thio)triphosphate-activated G_i3 and G_i2 compared with guanosine 5'-O-(β-thio)diphosphate-inactivated proteins. Similarly, in co-immunoprecipitation studies in HEK-293 cells, FLAG-tagged RASA3 preferentially interacted with activated mutants of G_i3 and G_i2 compared with wild type proteins. In GH4ZR7 cells, co-immunoprecipitation studies of endogenous proteins demonstrated a G_i3-RASA3 complex that was induced upon TRH/D2S receptor co-activation. To address RASA3 function in dopamine D2S receptor-induced inhibition of ERK1/2 activity, endogenous RASA3 protein expression was suppressed (70% knockdown) in GH4ZR7 cells stably transfected with full-length antisense cDNA of RASA3. The selected antisense clones had similar levels of dopamine D2S receptor binding and D2S-induced inhibition of cAMP formation compared with parental GH4ZR7 cells. In these clones, D2S-mediated inhibition of TRH-induced phospho-ERK1/2 was reversed by 70-80% compared with parental GH4ZR7 cells. Our results provide a novel mechanism for dopamine D2S-induced inhibition of ERK1/2 and indicate that RASA3 links G_i proteins to inhibit G_q-induced Ras/ERK1/2 activation.

Received for publication, May 12, 2008 , and in revised form, September 17, 2008.

^* This work was supported by grants from the Canadian Institutes of Health and the Ontario Mental Health Research (to P. R. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ Supported by Ontario Graduate Scholarship.

² Canadian Institutes of Health Research/Novartis Michael Smith Chair in Neurosciences. To whom correspondence should be addressed. Tel.: 613-562-5800, Ext. 8307; Fax: 613-562-5403; E-mail: palbert{at}uottawa.ca.

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