Heterotrimeric G-protein signaling systems are activated via cell surface receptors possessing the sevenmembrane span motif. Several observations suggest the existence of other modes of stimulus input to heterotrimeric G-proteins. As part of an overall effort to identify such proteins we developed a functional screen based upon the pheromone response pathway in Saccharomyces cerevisiae. We identified two mammalian proteins, AGS2 and AGS3 (activators of G-protein signaling), that activated the pheromone response pathway at the level of heterotrimeric G-proteins in the absence of a typical receptor. ␤-galactosidase reporter assays in yeast strains expressing different G␣ subunits (Gpa1, G s ␣, G i ␣ 2 (Gpa1(1-41)) , G i ␣ 3(Gpa1(1-41)) , G␣ 16(Gpa1(1-41)) ) indicated that AGS proteins selectively activated G-protein heterotrimers. AGS3 was only active in the G i ␣ 2 and G i ␣ 3 genetic backgrounds, whereas AGS2 was active in each of the genetic backgrounds except Gpa1. In protein interaction studies, AGS2 selectively associated with G␤␥, whereas AGS3 bound G␣ and exhibited a preference for G␣GDP versus G␣GTP␥S. Subsequent studies indicated that the mechanisms of G-protein activation by AGS2 and AGS3 were distinct from that of a typical G-proteincoupled receptor. AGS proteins provide unexpected mechanisms for input to heterotrimeric G-protein signaling pathways. AGS2 and AGS3 may also serve as novel binding partners for G␣ and G␤␥ that allow the subunits to subserve functions that do not require initial heterotrimer formation.The seven-membrane span hormone receptor coupled to heterotrimeric G-proteins represents one of the most widely used systems for information transfer across the cell membrane. Signal processing via this system likely operates within the context of a signal transduction complex. Within such a signal transduction complex, there are likely accessory proteins (distinct from receptor, G-protein, and effectors) that participate in the formation of this complex and/or regulate signal transfer from receptor to G-protein. In addition, several reports suggest alternative modes of stimulus input to heterotrimeric G-proteins that do not require direct interaction of the G-protein with the seven-membrane span receptor itself. To identify such entities and to define putative components of such a signal transduction complex we initiated two broad experimental approaches (1-4). One strategy focused on a functional readout involving G-protein activation and was based upon initial observations in our laboratory concerning the transfer of signal from R to G (3, 4). This approach resulted in the partial purification and characterization of the NG10815 G-protein activator that directly increased GTP␥S binding to brain G-protein in the absence of a receptor. To extend this body of work, we developed an expression cloning system in Saccharomyces cerevisiae that was designed to detect mammalian activators of the pheromone response pathway in the absence of a G-proteincoupled receptor (5). The pheromone response pathw...

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 Gprotein. 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 DDT 1 -MF2. Immunofluorescence studies and confocal imaging indicated that AGS3 was predominantly cytoplasmic and enriched in microdomains of the cell. AGS3 coimmunoprecipitated with G␣ i3 from cell and tissue lysates, indicating that a subpopulation of AGS3 and G␣ i exist as a complex in the cell. The coimmunoprecipitation of AGS3 and G␣ i was dependent upon the conformation of G␣ i3 (GDP > > GTP␥S (guanosine 5-3-O-(thio)triphosphate)). The regions of AGS3 that bound G␣ i were localized to four amino acid repeats (G-protein regulatory motif (GPR)) in the carboxyl terminus (Pro 463 -Ser 650 ), each of which were capable of binding G␣ i . AGS3-GPR domains selectively interacted with G␣ i in tissue and cell lysates and with purified G␣ i /G␣ t . Subsequent experiments with purified G␣ i2 and G␣ i3 indicated that the carboxyl-terminal region containing the four GPR motifs actually bound more than one G␣ i subunit at the same time. The AGS3-GPR domains effectively competed with G␤␥ for binding to G␣ t(GDP) and blocked GTP␥S binding to G␣ i1 . AGS3 and related proteins provide unexpected mechanisms for coordination of G-protein signaling pathways.Signal processing via heterotrimeric G-protein proteins generally involves an initial input sensed by a cell surface receptor with seven membrane-spanning regions. Conformational changes in receptor subdomains then transfer this signal to a G-protein, promoting exchange of GTP for GDP and subunit dissociation with both the G␣ and G␤␥ subunits regulating effector molecules. These events are tightly regulated to maximize signal efficiency, optimize signal specificity, and integrate cellular responses to diverse stimuli. Regulatory mechanisms include the segregation of specific signaling molecules in cell microdomains, receptor phosphorylation and internalization, cross-talk between signaling pathways, and proteins that regulate the basal activation state of G-proteins independently of the receptor.We partially purified a direct G-protein activator from NG108-15 cells (1, 2) and subsequently used a functional screen to identify three proteins (AGS1-3, for activator of Gprotein signaling 1-3) that activated heterotrimeric G-protein signaling in the absence of a cell surface receptor (3-5). The identification of such proteins raises many interesting and unexpected questions relative to signal processing by heterotrimeric G-proteins. As an initial approach to address these issues, we focused on the biochemical and functional characterizati...

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The G-protein regulatory (GPR) motif in AGS3 was recently identified as a region for protein binding to heterotrimeric G-protein ␣ subunits. To define the properties of this ϳ20-amino acid motif, we designed a GPR consensus peptide and determined its influence on the activation state of G-protein and receptor coupling to G-protein. The GPR peptide sequence (28 amino acids) encompassed the consensus sequence defined by the four GPR motifs conserved in the family of AGS3 proteins. The GPR consensus peptide effectively prevented the binding of AGS3 to Gi␣1,2 in protein interaction assays, inhibited guanosine 5-O-(3-thiotriphosphate) binding to Gi␣, and stabilized the GDP-bound conformation of Gi␣. The GPR peptide had little effect on nucleotide binding to Go␣ and brain G-protein indicating selective regulation of Gi␣. Thus, the GPR peptide functions as a guanine nucleotide dissociation inhibitor for Gi␣. The GPR consensus peptide also blocked receptor coupling to Gi␣␤␥ indicating that although the AGS3-GPR peptide stabilized the GDP-bound conformation of Gi␣, this conformation of Gi␣ GDP was not recognized by a G-protein coupled receptor. The AGS3-GPR motif presents an opportunity for selective control of Gi␣-and G␤␥؊regulated effector systems, and the GPR motif allows for alternative modes of signal input to G-protein signaling systems.The G-protein regulatory (GPR) 1 motif or GoLOCO repeat is a ϳ20-amino acid domain found in several proteins that interact with and/or regulate G-proteins (1, 2). Such proteins include the activator of G-protein signaling AGS3, the AGS3-related protein PINS in Drosophila melanogaster, two members of the RGS family of proteins, and three proteins (LGN, Pcp2, and Rap1GAP) isolated in yeast two-hybrid screens using Gi␣ or Go␣ as bait. Rat AGS3 was isolated in a yeast-based functional screen designed to identify receptorindependent activators of heterotrimeric G-protein signaling (1). The AGS3-related protein PINS is required for asymmetric cell division of neuroblasts in D. melanogaster, where it is found complexed with Gi/Go (3, 4), but neither the signal input nor output for this complex is known. Some insight as to how PINS may regulate Gi/Go is provided by studies with AGS3 (1). In the yeast-based system, AGS3 selectively activated Gi␣2 and Gi␣3. The action of AGS3 as a G-protein activator in the yeast-based system was independent of nucleotide exchange as it was not antagonized by overexpression of RGS4, and it was still observed following replacement of Gi␣2 with Gi␣2-G204A, a mutant that is deficient in making the transition to the GTP-bound state (1, 5). Both of these manipulations effectively prevent receptor-mediated activation of G-protein signaling in the yeast system and block the action of AGS1, which was isolated in the same screen and apparently behaves as a guanine nucleotide exchange factor for heterotrimeric G-proteins (5, 6). These data indicate that the interaction of AGS3 with G-protein influences a unique control mechanism within the activation/deactivation cycle...

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