GAP1(IP4BP)RASA3 mediates Galpha(i)-induced inhibition of mitogen-activated protein kinase in pituitary cells

Abstract

Activation of dopamine D2S receptor (short isoform) inhibits thyrotropin-releasing hormone (TRH)-stimulated p42/p44 mitogen-activated protein kinase (MAPK) phosphorylation in rat pituitary GH4ZR7 cells via Galphai3, but the underlying mechanism is not fully understood. To identify novel Galphai3 effectors we performed a yeast two-hybrid screen on cDNA library from GH4ZR7 cells using constitutively active Galphai3 as bait and identified RASA3. Galphai3-RASA3 interactions were confirmed using different approaches including yeast mating/beta-galactosidase assay, in vitro pull-down assay on bacterially expressed proteins, and co-immunoprecipitation with overexpressed or endogenous proteins in different mammalian cell lines. To address RASA3 function in dopamine D2S receptor-induced inhibition of MAPK activity endogenous RASA3 was suppressed in GH4ZR7 cells. In these clones D2S-mediated inhibition of TRH-induced phospho-MAPK was reversed by 70-80% compared to parental cells. Our results provide a novel mechanism for D2S-induced inhibition of MAPK and indicate that RASA3 links Galphai3 to inhibition of the powerful TRH-induced Ras/MAPK activation pathway.