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...

Our results suggest that GPR-1 and/or GPR-2 control an asymmetry in forces exerted on the spindle poles by asymmetrically modulating the activity of the heterotrimeric G protein in response to a signal from the PAR proteins.

We describe genetic screens in Saccharomyces cerevisiae designed to identify mammalian nonreceptor modulators of G-protein signaling pathways. Strains lacking a pheromone-responsive G-protein coupled receptor and expressing a mammalian-yeast Galpha hybrid protein were made conditional for growth upon either pheromone pathway activation (activator screen) or pheromone pathway inactivation (inhibitor screen). Mammalian cDNAs that conferred plasmid-dependent growth under restrictive conditions were identified. One of the cDNAs identified from the activator screen, a human Ras-related G protein that we term AGS1 (for activator of G-protein signaling), appears to function by facilitating guanosine triphosphate (GTP) exchange on the heterotrimeric Galpha. A cDNA product identified from the inhibitor screen encodes a previously identified regulator of G-protein signaling, human RGS5.

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...

Utilizing a functional screen in the yeast Saccharomyces cerevisiae we identified mammalian proteins that activate heterotrimeric G-protein signaling pathways in a receptor-independent fashion. One of the identified activators, termed AGS1 (for activator of G-protein signaling), is a human Ras-related G-protein that defines a distinct subgroup of the Ras superfamily. Expression of AGS1 in yeast and in mammalian cells results in specific activation of G␣ i /G␣ o heterotrimeric signaling pathways. In addition, the in vivo and in vitro properties of AGS1 are consistent with it functioning as a direct guanine nucleotide exchange factor for G␣ i /G␣ o . AGS1 thus presents a unique mechanism for signal integration via heterotrimeric G-protein signaling pathways. GPCR1 signaling pathways represent one of the most widely used mechanisms in nature for transducing signals from the extracellular to the intracellular environment. Each step in the activated GPCR signaling cascade presents a potential regulatory checkpoint for fine-tuning and directing the signal. Although a number of regulatory molecules affecting GPCR signaling have been identified (1)(2)(3)(4)(5)(6)(7)(8), evidence suggests the presence of additional pathway modulators (8 -10). To isolate such modulators, we developed a series of functional screens in the yeast Saccharomyces cerevisiae designed to detect mammalian proteins that either activate or inactivate the pheromone response pathway, a G-protein coupled pathway in which G␤␥ acts as the positive signal transducer (11,12). Genetic manipulation of the yeast strains allowed detection of mammalian modulators through simple growth screens, and the functional redundancy between the pheromone response pathway and mammalian GPCR pathways (13-16) allowed us to replace the yeast G␣ with human G␣ i2 , thereby biasing the screens toward the non-yeast component of the pathway. From these screens we identified three mammalian proteins that appeared to activate signaling by distinct mechanisms (11,12). As expression of these proteins did not alter G-protein expression levels in yeast, we termed these proteins AGS for activators of G-protein signaling. This report describes the functional characterization of AGS1, a Ras-related protein isolated from a screen of human liver cDNA. EXPERIMENTAL PROCEDURESStrains and Plasmids-Plasmid constructions, except as indicated below, have been described previously (11). Plasmid pSV-␤gal was purchased from Promega; pYES2, pCEP4, pcDNA3.1(ϩ), pcDNA3.1-His-lacZ, and pcDNA3.1-HisC were from Invitrogen; pYEX4T1 was from Amrad Biotech and pFA2-cJun, pFA2-Elk1, pFA2-CREB, pFA-CHOP, pFR-Luc, pFC-MEK1, and pBluescriptSK(ϩ) were from Stratagene. A plasmid carrying human transducin-␣ (GNAZ) cDNA sequences in pBluescriptSK(ϩ) was a gift from M. Simon. AGS1 and AGS1-G31V (11) were amplified from pYES2 plasmids and ligated into pcDNA3.1-HisC and pYEX4T1, placing the AGS1 coding sequences in-frame with, respectively, an N-terminal His 6 tag sequence and an N-terminal GST sequence. In a similar f...