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J. Biol. Chem., Vol. 267, Issue 4, 2364-2369, 02, 1992

Translocation of alpha subunits of stimulatory guanine nucleotide- binding proteins through stimulation of the prostacyclin receptor in mouse mastocytoma cells

M Negishi, H Hashimoto and A Ichikawa
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Japan.

The translocation of the alpha subunits of Gs from the membrane to the cytosol by iloprost, a stable prostacyclin analogue, was studied in mouse mastocytoma P-815 cells. In the presence of guanosine 5'-O- (thiotriphosphate) (GTP gamma S), iloprost stimulated the adenylate cyclase activity, caused the release of both 42- and 45-kDa proteins reactive with the anti Gs alpha carboxyl-terminal antibody, RM/1, from the membrane and attenuated cholera toxin-catalyzed ADP-ribosylation of the 42- and 45-kDa proteins in the membrane. The iloprost-stimulated adenylate cyclase activity and release of Gs alpha from the membrane were markedly suppressed by RM/1. Cholera toxin treatment also stimulated the adenylate cyclase activity and release of Gs alpha from the membrane, and iloprost synergistically potentiated these actions of cholera toxin. In mastocytoma cells, iloprost induced the translocation of both 42- and 45-kDa Gs alpha from the membrane to the cytosol, 45- kDa Gs alpha remaining in the cytosol for a longer time than 42- kDa Gs alpha. Whereas 42-kDa Gs alpha in the cytosol was eluted at the position of Mr = approximately 40,000 45-kDa Gs alpha was eluted at the position of Mr = approximately 120,000 from a Superose 12 gel filtration column. In contrast, both 42- and 45-kDa Gs alpha released in vitro from the membrane by iloprost plus GTP gamma S were eluted at the position of Mr = approximately 40,000, but only 45-kDa Gs alpha was eluted at the position of Mr = approximately 120,000 when it was incubated with cytosol. These results taken together demonstrate that iloprost induces the translocation of both 42- and 45-kDa Gs alpha from the membrane to the cytosol and that only the 45-kDa Gs alpha released exists in the cytosol as a soluble complex with unidentified component(s) in mastocytoma cells.
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