Activation of Heterotrimeric G-protein Signaling by a Ras-related Protein

Abstract

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...

The small GTP-binding protein, Rhes, regulates signal transduction from G protein-coupled receptors

“…These results strongly suggest that Rhes is acting somewhere upstream of the activation of the heterotrimeric complex by the receptor. This would be in agreement with data from Cismowski et al (1999Cismowski et al ( , 2000, Takesono et al (2002), and Graham et al (2002) in which a Ras family protein would directly modulate the activation of a heterotrimeric G protein. The mechanism is not clear.…”

“…Recent evidence implicates the Rhes homolog Dexras1 in the activation of Gai proteins (Cismowski et al, 2000). Indeed, a Dexras cDNA clone was isolated in a screen designed to identify proteins promoting the emission of signals by heterotrimeric G proteins.…”

“…Among the most recent additions made to the Ras superfamily are two proteins whose expression is regulated by hormones, Dexras1 and Rhes: Dexras1 was originally found in mice, and its name reflects the fact that it was induced by dexamethasone (Kemppainen and Behrend, 1998). A human Dexras homologue was subsequently isolated in a functional screen in yeast designed to identify receptor-independent activators of heterotrimeric G protein signaling, and was named as AGS1 (Cismowski et al, 1999(Cismowski et al, , 2000. AGS1 was proposed to function as a guanine nucleotide exchange factor for Gai/Ga0.…”

The Ras-related protein AGS1/RASD1 suppresses cell growth

“…Similar inhibition of colony formation was observed with the human His-tagged AGS1/RASD1 ( Figure 2). AGS1-G31V, which contains a mutation within the PM1 domain of the Ras core and exhibited a reduced activity in both the yeast functional screen and Elk activation assays in COS-7 transfectants Cismowski et al, 2000), did not alter the number of G418-resistant colonies, providing an additional internal control for the specificity of the observed effects of wild-type AGS1/RASD1. Expansion of colonies that did grow in the AGS1/RASD1-transfected plate followed by immunoblotting indicated that, although G418-resistant, these cells did not express detectable levels of AGS1/RASD1 (Vaidyanathan G and Lanier SM.…”

“…AGS1/RASD1 was first discovered as a dexamethasone-inducible cDNA (Dexras1) in AtT-20 mouse corticotroph cells (Kemppainen and Behrend, 1998) and subsequently as a receptor-independent activator of heterotrimeric G-protein signaling (hence its name Activator of G-protein Signaling) in a yeastbased functional screen of mammalian cDNAs Takesono et al, 1999). As both AGS1/RASD1 and Rhes/TEM2/RASD2 can influence signaling pathways involving heterotrimeric G-proteins (Cismowski et al, , 2000Graham et al, 2001Graham et al, , 2002Takesono et al, 2002;Vargiu et al, 2004), they provide interesting points for signal integration and cross-talk. AGS1/RASD1 is also involved in other signaling pathways as it forms an apparent ternary complex with neuronal nitric oxide synthase (nNOS) and the nNOS binding protein CAPON, where the activation of AGS1/ RASD1 is suggested to involve S-nitrosylation (Fang et al, 2000;Jaffrey et al, 2002).…”