Signal-activated G protein-coupled receptors (GPCRs) 1 stimulate GDP/GTP exchange on the ␣ subunits of G proteins. Following the activational interaction with receptors, G␣GTP and G␤␥ are released to activate their targets, which include adenylyl cyclases, phospholipases, phosphodiesterases, and ion channels (1-3). A novel class of GTPase-activating proteins (GAPs) for G proteins termed regulators of G protein signaling (RGS) has been identified (4 -6). RGS proteins share a highly conserved RGS domain, which is responsible for the GAP function. Recently, cloning of proteins critical for glial cell development resulted in the identification of the first known Drosophila RGS protein, LOCO (7). The LOCO sequence revealed significant homology to RGS12 and RGS14 within the RGS domain and three additional regions B, C, and D (7). A yeast two-hybrid screen was carried out using G i ␣ as bait in an attempt to confirm the interaction of LOCO with G␣. Interestingly, the D region, rather than the RGS domain of LOCO, was found to bind G i ␣ (7). Sequence analysis of the D region revealed that it contained a segment of homology with four ϳ20-amino acid repeats present in the human mosaic protein, LGN.LGN has been previously identified as a G i ␣ 2 -interacting protein using a yeast two-hybrid system (8).LGN is similar to the activator of G protein signaling 3 (AGS3), which was isolated in a functional screen for receptor-independent activators of heterotrimeric G protein signaling (9). Site-directed mutagenesis and protein interaction studies with AGS3 (9) indicated that the ϳ20-amino acid repeats common to AGS3, LGN, and LOCO were responsible for binding G i ␣. The ϳ20-amino acid repeats were termed the G protein regulatory (GPR) (9) or GoLOCO motif (10). The GPR motif was also identified in Purkinje cell protein-2 (Pcp2) and Rap1GAP, which were identified as G o ␣ binding partners in yeast-two hybrid screens (11,12). These studies suggest that GPR-containing proteins, hereafter termed GPR proteins, are likely to represent a diverse family of proteins that modulate G protein signaling. At present very little is known about the mechanisms and functions of GPR proteins. The yeast pheromone response pathway is mediated by G␤␥ subunits, and its GPCR-independent activation by AGS3 suggests that it may induce release of G␤␥ from G proteins (9). Pcp2 protein was shown to stimulate GDP release from G o ␣ without affecting the k cat for GTP hydrolysis, thus raising the possibility that GPR proteins may serve as guanine nucleotide exchange factors for G proteins (11). To date, no studies on the regulation of GPCR-mediated G protein activation by GPR proteins have been reported. In this study, we examined the effects of the AGS3 GPR domain (AGS3GPR) on the intrinsic guanine nucleotide exchange of G i ␣