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.

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...

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