Selective interaction of AGS3 with G-proteins and the influence of AGS3 on the activation state of G-proteins

Abstract

AGS3 (activator of G-protein signaling 3) was isolated in a yeast-based functional screen for receptor-independent activators of heterotrimeric G-proteins. As an initial approach to define tile role of AGS3 in mammalian signal processing, we defined the AGS3 subdomains involved in G-protein interaction, its selectivity for G-proteins, and its influence on the activation state of G-protein. Immunoblot analysis with AGS3 antisera indicated expression in rat brain, the neuronal-like cell lines PC12 and NG108-15, as well as the smooth muscle cell line DDT1-MF2. Immunofluorescence studies and confocal imaging indicated that AGS3 was predominantly cytoplasmic and enriched in microdomains of the cell. AGS3 coimmunoprecipitated with Gαi3 from cell and tissue lysates, indicating that a subpopulation of AGS3 and Gαi exist as a complex in the cell. The coimmunoprecipitation of AGS3 and Gαi3 was dependent upon the conformation of Gαi3 (GDP ≫ GTPγS (guanosine 5′-3-O-(thio)triphosphate)). The regions of AGS3 that bound Gαi were localized to four amino acid repeats (G-protein regulatory motif (GPR)) in the carboxyl terminus (Pro463-Ser650), each of which were capable of binding Gαi. AGS3-GPR domains selectively interacted with Gαi in tissue and cell lysates and with purified Gαi/Gαt. Subsequent experiments with purified Gα12 and Gα13 indicated that the carboxyl-terminal region containing the four GPR motifs actually bound more than one Gαi subunit at the same time. The AGS3-GPR domains effectively competed with Gβγ for binding to Gαt(GDP) and blocked GTPγS binding to Gαi1. AGS3 and related proteins provide unexpected mechanisms for coordination of G-protein signaling pathways.