AGS3 inhibits GDP dissociation from Gα subunits of the Gi family and rhodopsin-dependent activation of transducin

A number of recently discovered proteins that interact with the alpha subunits of G(i)-like G proteins contain homologous repeated sequences named G protein regulatory (GPR) motifs, Activator of G: protein signaling 3 (AGS3), identified as an activator of the yeast pheromone pathway in the absence of the pheromone receptor, has a domain with four such repeats. To elucidate the potential mechanisms of regulation of G protein signaling by proteins containing GPR motifs, we examined the effects of the AGS3 GPR domain on the kinetics of guanine nucleotide exchange and GTP hydrolysis by G(i)alpha (1) and transducin-alpha (G(t)alpha). The AGS3 GPR domain markedly inhibited the rates of spontaneous guanosine 5'-O-(3-thiotriphosphate) (GTP gammaS) binding to G(i)alpha and rhodopsin-stimulated GTP gammaS binding to G(i)alpha The full-length AGS3 GPR domain, AGS3-(463-650), was similar to 30-fold more potent than AGS3-(572-629), containing two AGS3 GPR motifs, The IC50 values for the AGS3-(463-650) inhibitory effects on G(i)alpha and transducin were 0.12 and 0.15 muM, respectively. Furthermore, AGS3-(463-650) and AGS3-(572-629) effectively blocked the GDP release from G(i)alpha and rhodopsin-induced dissociation of GDP from G(i)alpha The potencies of AGS3-(572-629) and AGS3(463-650) to suppress the GDP dissociation rates correlated with their ability to inhibit the rates of GTP gammaS binding. Consistent with the inhibition of nucleotide exchange, the AGS3 GPR domain slowed the rate of steady-state GTP hydrolysis by G(i)alpha The catalytic rate of G(i)alpha GTP hydrolysis, measured under single turnover conditions, remained unchanged with the addition of AGS3-(463-650). Altogether, our results suggest that proteins containing GPR motifs, in addition to their potential role as G protein-coupled receptor-independent activators of G beta gamma signaling pathways, act as GDP dissociation inhibitors and negatively regulate the activation of a G protein by a G protein-coupled receptor.