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...

Uridine 5-diphosphoglucose (UDP-glucose) has a well established biochemical role as a glycosyl donor in the enzymatic biosynthesis of carbohydrates. It is less well known that UDP-glucose may possess pharmacological activity, suggesting that a receptor for this molecule may exist. Here, we show that UDP-glucose, and some closely related molecules, potently activate the orphan G protein-coupled receptor KIAA0001 heterologously expressed in yeast or mammalian cells. Nucleotides known to activate P2Y receptors were inactive, indicating the distinctly novel pharmacology of this receptor. The receptor is expressed in a wide variety of human tissues, including many regions of the brain. These data suggest that some sugar-nucleotides may serve important physiological roles as extracellular signaling molecules in addition to their familiar role in intermediary metabolism.

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...

Neuropeptides are a structurally diverse group of hormones and neurotransmitters that bind to a related subfamily of G protein-coupled receptors (1) and function in neuron-to-neuron communication, as well as in signaling in the immune system and in tissue restructuring. Neuropeptides and their receptors are the principal driving force behind one of the most clinically aggressive cancers, small cell lung cancer (SCLC).1 SCLC tumors sustain their growth, in part, by maintaining neuropeptide autocrine and paracrine loops (2). These tumor cells in culture can secrete and respond mitogenically to multiple neuropeptides (3). (4) can not only inhibit the action of substance P (5) but also inhibit binding and action of gastrin-releasing peptide (GRP), arginine vasopressin (6), and endothelin (7). To date, much of the research on the mechanism of action of SPDs has focused on their abilities to block ligand binding and Ca 2ϩ flux, as well as on their cytostatic or cytotoxic effects on SCLC cells in culture (8).The signal transduction pathways that mediate neuropeptide actions are rapidly becoming more clear. It is known that neuropeptides induce calcium mobilization by a pertussis toxin-insensitive mechanism, suggesting a role for G q (9). Heterologous expression of receptors and G proteins in Sf9 cells have shown functional coupling between one neuropeptide receptor, NK-1, and G z , G q , and G 13 but not G s (10). In addition to the mobilization of Ca 2ϩ from intracellular stores, neuropeptides can have diverse effects on cells in culture, including the induction of mitogenesis (11), the activation of both the extracellular signal-regulated kinase (ERK) (12) and the c-Jun Nterminal kinase (JNK) (13, 14) members of the mitogenactivated protein kinase family, and formation of actin structures such as filopodia (15) and stress fibers (16,17). The activation of JNK and the reorganization of the actin cytoskeleton are most likely mediated by members of the G 12 family of G proteins, such as G␣ 12 and G␣ 13 (18,19). The regulation of ERKs by neuropeptides is more complicated, suggesting a role for PKC (20), G protein ␤␥ subunits (12), and a pertussis toxin-sensitive mechanism possibly involving a G i family member (21).The effects of SPD-D and similar compounds on these signaling events downstream of neuropeptide receptors suggest a mechanism that is more complicated than that of a classical antagonist. The ERK response and the Ca 2ϩ response through neuropeptide receptors are affected differently by 23). In Swiss 3T3 fibroblasts, SPD-D inhibits Ca 2ϩ mobilization induced by bombesin (which acts on the human GRP receptor) with a maximal effect at 10 M and an estimated IC 50 of 2 M. In contrast, SPD-D inhibits ERK-2 activation with a maximal effect at 50 M and an estimated IC 50 of 9 M (22). In the presence of 3 nM bombesin, 10 M SPD-D caused a nearly complete inhibition of the Ca 2ϩ response and an approximately 50% inhibition of the ERK-2 response (22). In a similar manner, 50 M SPD-D caused a 100% inhibition of the inosit...