Regulators of G protein signaling (RGS) act as GTPase-activating proteins for Galpha(i) and for Galpha(q/11). There is recent evidence for interaction of RGS proteins with Galpha(s), and substitution of Ser for Asp(229) in RGS proteins enhances interactions with G proteins. Site-directed mutagenesis of Asp(229) was used to assess the effect of this site on the gonadotropin-releasing hormone receptor (GnRHR)-Galpha(s) mediated signaling in the absence or presence of over-expressed RGS3, RGS10 or a truncated form of RGS3 (RGS3T). We observed increased cAMP release with the mutant Galpha(s)(D(229)S) compared to wt Galpha(s) when GGH(3) cells (GH(3) cells stably expressing the GnRH receptor) were stimulated with a GnRH agonist. In the presence of RGS3, we did not observe any difference in cAMP release with wt Galpha(s) or with Galpha(s)(D(229)S) compared to control values; in the presence of RGS3T there was an increase of cAMP release with wt Galpha(s) compared to the control but there was no difference between the Galpha(s)(D(229)S) and the control values. When cells co-expressed wt Galpha(s) and RGS10, there was a significant increase of cAMP release compared with cells co-expressing wt Galpha(s) and Lac Z. Cells co-expressing Galpha(s)(D(229)S) and RGS10 showed a significant increase of cAMP release compared to control cells. These results indicate differential regulation of the GnRHR-Galpha(s) mediated signaling by a single mutation in Galpha(s) in the presence of RGS10 and RGS3T, but not with RGS3. This is the first report of an effect of the Galpha(s)(D(229)S) mutation on G protein-coupled receptor-mediated activation.