Receptor-independent activators of heterotrimeric G-proteins.

Heterotrimeric G-protein signalling systems are primarily activated via cell surface receptors possessing the seven membrane span motif. Several observations suggest the existence of other modes of input to such signalling systems either downstream of effectors or at the level of G-proteins themselves. Using a functional screen based upon the pheromone response pathway in Saccharomyces cerevisiae, we identified three proteins, AGS1-3 (for Activators of G-protein Signalling), that activated heterotrimeric G-protein signalling pathways in the absence of a typical receptor. AGS1 defines a distinct member of the super family of ras related proteins. AGS2 is identical to mouse Tctex1, a protein that exists as a light chain component of the cytoplasmic motor protein dynein and subserves as yet undefined functions in cell signalling pathways. AGS3 possesses a series of tetratrico repeat motifs and a series of four amino acid repeats termed G-protein regulatory motifs. The GPR motifs are found in a number of proteins that interact with and regulate Galpha. Although each AGS protein activates G-protein signaling, they do so by different mechanisms within the context of the G-protein activation/deactivation cycle. AGS proteins provide unexpected mechanisms for input to heterotrimeric G-protein signalling pathways.

Identification of a truncated form of the G-protein regulator AGS3 in heart that lacks the tetratricopeptide repeat domains.

AGS3, a 650-amino acid protein encoded by an approximately 4-kilobase (kb) mRNA enriched in rat brain, is a Galpha(i)/Galpha(t)-binding protein that competes with Gbetagamma for interaction with Galpha(GDP) and acts as a guanine nucleotide dissociation inhibitor for heterotrimeric G-proteins. An approximately 2-kb AGS3 mRNA (AGS3-SHORT) is enriched in rat and human heart. We characterized the heart-enriched mRNA, identified the encoded protein, and determined its ability to interact with and regulate the guanine nucleotide-binding properties of G-proteins. Screening of a rat heart cDNA library, 5'-rapid amplification of cDNA ends, and RNase protection assays identified two populations of cDNAs (1979 and 2134 nucleotides plus the polyadenylation site) that diverged from the larger 4-kb mRNA (AGS3-LONG) in the middle of the protein coding region. Transfection of COS-7 cells with AGS3-SHORT cDNAs resulted in the expression of a major immunoreactive AGS3 polypeptide (M(r) approximately 23,000) with a translational start site at Met(495) of AGS3-LONG. Immunoblots indicated the expression of the M(r) approximately 23,000 polypeptide in rat heart. Glutathione S-transferase-AGS3-SHORT selectively interacted with the GDP-bound versus guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-bound conformation of Galpha(i2) and inhibited GTPgammaS binding to Galpha(i2). Protein interaction assays with glutathione S-transferase-AGS3-SHORT and heart lysates indicated interaction of AGS3-SHORT with Galpha(i1/2) and Galpha(i3), but not Galpha(s) or Galpha(q). Immunofluorescent imaging and subcellular fractionation following transient expression of AGS3-SHORT and AGS3-LONG in COS-7 and Chinese hamster ovary cells indicated distinct subcellular distributions of the two forms of AGS3. Thus, AGS3 exists as a short and long form, both of which apparently stabilize the GDP-bound conformation of Galpha(i), but which differ in their tissue distribution and trafficking within the cell.

Receptor-independent activators of heterotrimeric G-protein signaling pathways.

Heterotrimeric G-protein signaling systems are activated via cell surface receptors possessing the seven-membrane 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. beta-galactosidase reporter assays in yeast strains expressing different Galpha subunits (Gpa1, G(s)alpha, G(i)alpha(2(Gpa1(1-41))), G(i)alpha(3(Gpa1(1-41))), Galpha(16(Gpa1(1-41)))) indicated that AGS proteins selectively activated G-protein heterotrimers. AGS3 was only active in the G(i)alpha(2) and G(i)alpha(3) genetic backgrounds, whereas AGS2 was active in each of the genetic backgrounds except Gpa1. In protein interaction studies, AGS2 selectively associated with Gbetagamma, whereas AGS3 bound Galpha and exhibited a preference for GalphaGDP versus GalphaGTPgammaS. Subsequent studies indicated that the mechanisms of G-protein activation by AGS2 and AGS3 were distinct from that of a typical G-protein-coupled 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 Galpha and Gbetagamma that allow the subunits to subserve functions that do not require initial heterotrimer formation.

Negative regulation of alpha2-adrenergic receptor-mediated Gi signalling by a novel pathway.

Chinese hamster ovary (CHO) cells stably expressing alpha(2) adrenergic receptor (alpha(2)AR) were pretreated with cholera toxin (CTX) and then treated with or without PMA. The alpha(2A)AR-mediated inhibition of forskolin-stimulated cAMP accumulation was completely ablated by CTX pretreatment only after additional treatment with PMA. Although the addition of cycloheximide (protein synthesis inhibitor) and H-89 (cAMP dependent protein kinase inhibitor) did not completely counteract the negative regulation, the elevation of cAMP was a primary factor for negative regulation by treatment with CTX and PMA. In contrast with the cAMP response, the inhibition of membrane adenylate cyclase activity and the agonist competition curve were not influenced by treatment with CTX or PMA, suggesting that a cytosolic factor was involved in this negative regulation. The m2-muscarinic-acetylcholine-receptor-mediated inhibition of the forskolin-stimulated accumulation of cAMP was also attenuated by treatment with CTX and PMA. The ablation of alpha(2A)AR-mediated inhibition was not observed when alpha(2A)AR was expressed in Rat2 fibroblast cells, suggesting that this negative regulation is not dependent on the receptor type but is instead a phenomenon common to G(i)-coupled receptors in CHO cells. Reverse-transcriptase-mediated PCR and Northern blot analysis showed that the expression of GOS8/RGS2 mRNA, which is a member of the regulator of G-protein signalling (RGS) group of proteins, was considerably increased by pretreatment with CTX. These results indicate a novel regulatory pathway, whereby a cytosolic factor induced by the elevation of cellular cAMP levels negatively regulates G(i) signalling in a protein-kinase-C-dependent manner.

Genetic screens in yeast to identify mammalian nonreceptor modulators of G-protein signaling.

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.