AGS3 inhibits GDP dissociation from G alpha subunits of the G(i) family and rhodopsin-dependent activation of transducin

A number of recently discovered proteins that interact with the alpha subun its of G(i)-like G proteins contain homologous repeated sequences named G p rotein regulatory (GPR) motifs, Activator of G: protein signaling 3 (AGS3), identified as an activator of the yeast pheromone pathway in the absence o f 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 th e 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)alp ha 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 v alues 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 t heir ability to inhibit the rates of GTP gammaS binding. Consistent with th e 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 G TP hydrolysis, measured under single turnover conditions, remained unchange d with the addition of AGS3-(463-650). Altogether, our results suggest that proteins containing GPR motifs, in addition to their potential role as G p rotein-coupled receptor-independent activators of G beta gamma signaling pa thways, act as GDP dissociation inhibitors and negatively regulate the acti vation of a G protein by a G protein-coupled receptor.