GAP1 (IP4BP)/RASA3 Mediates Gαi-induced Inhibition of Mitogen-activated Protein Kinase.

The dopamine D2S receptor (short isoform) couples to inhibitory Gαi/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 Gαi effectors, yeast two-hybrid screening of a GH4ZR7 cDNA library was done using constitutively active Gαi3-Q204L, and multiple clones of the RasGAP cDNA GAP1IP4BP/RASA3 were identified. In yeast mating assay, RASA3 preferentially interacted with activated forms of Gαi/o/2 proteins, but not with Gas. A direct interaction was indicated by in vitro pull-down assay, in which S-His-RASA3 preferentially bound guanosine 5'-O-(γ-thio)triphosphate-activated Gαi3 and Gαi2 compared with guanosme 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 Gαi3 and Gαi2 compared with wild type proteins. In GH4ZR7 cells, co-immunoprecipitation studies of endogenous proteins demonstrated a Gαi3-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 Gαi proteins to inhibit Gq-induced Ras/ ERK1/2 activation. [ABSTRACT FROM AUTHOR]