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

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 the 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 G alpha (i3) from cell and tissue lysates, indicating that a subpopulation of AGS3 and G alpha (1) exist as a complex in the cell. The coimmunoprecipitation of AGS3 and G alpha (i) was dependent upon the conformation of G alpha (i3) (GDP much greater than GTP gammaS (guanosine 5'-3-O-(thio)triphosphate)). The regions of AGS3 that bound G alpha (i) were localized to four amino acid repeats (G-protein regulatory motif (GPR)) in the carboxyl terminus (Pro(465)-Ser(650)), each of which were capable of binding G alpha (i). AGS3-GPR domains selectively interacted with G alpha (i) in tissue and cell lysates and with purified G alphai/G(alphat). Subsequent experiments with purified G alpha (i2) and G alpha (i3) indicated that the carboxyl-terminal region containing the four GPR motifs actually bound more than one G alpha (i) subunit at the same time. The AGS3GPR domains effectively competed with GP gamma for binding to G alpha (t(GDP)) and blocked GTP gammaS binding to G alpha (i). AGS3 and related proteins provide unexpected mechanisms for coordination of G-protein signaling pathways.