Receptor-independent Activators of Heterotrimeric G-protein Signaling Pathways (1999)

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-proteinsignaling), that activated the pheromone response pathway at the level of heterotrimeric G-proteins in the absence of a typical receptor. β-galactosidase reporter assays in yeast strains expressing different Gα subunits (Gpa1, Gsα, Giα2(Gpa1(1–41)), Giα3(Gpa1(1–41)), Gα16(Gpa1(1–41))) indicated that AGS proteins selectively activated G-protein heterotrimers. AGS3 was only active in the Giα2 and Giα3genetic backgrounds, whereas AGS2 was active in each of the genetic backgrounds except Gpa1. In protein interaction studies, AGS2 selectively associated with Gβγ, whereas AGS3 bound Gα and exhibited a preference for GαGDP versus GαGTPγS. 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 Gα and Gβγ that allow the subunits to subserve functions that do not require initial heterotrimer formation. 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-proteinsignaling), that activated the pheromone response pathway at the level of heterotrimeric G-proteins in the absence of a typical receptor. β-galactosidase reporter assays in yeast strains expressing different Gα subunits (Gpa1, Gsα, Giα2(Gpa1(1–41)), Giα3(Gpa1(1–41)), Gα16(Gpa1(1–41))) indicated that AGS proteins selectively activated G-protein heterotrimers. AGS3 was only active in the Giα2 and Giα3genetic backgrounds, whereas AGS2 was active in each of the genetic backgrounds except Gpa1. In protein interaction studies, AGS2 selectively associated with Gβγ, whereas AGS3 bound Gα and exhibited a preference for GαGDP versus GαGTPγS. 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 Gα and Gβγ that allow the subunits to subserve functions that do not require initial heterotrimer formation. activators of G-protein signaling glutathione S-transferase guanosine 5′-3-O-(thio)triphosphate nucleotide(s) G-protein regulatory rapid amplification of cDNA ends 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 (1Wu G. Krupnick J.G. Benovic J.L. Lanier S.M. J. Biol. Chem. 1997; 272: 17836-17842Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 2Wu G. Hildebrandt J. Benovic J.L. Lanier S.M. J. Biol. Chem. 1998; 273: 7197-7200Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 3Sato M. Kataoka R. Dingus J. Wilcox M. Hildebrandt J. Lanier S.M. J. Biol. Chem. 1995; 270: 15269-15276Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 4Sato M. Ribas C. Hildebrandt J.D. Lanier S.M. J. Biol. Chem. 1996; 271: 30052-30060Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). 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 (3Sato M. Kataoka R. Dingus J. Wilcox M. Hildebrandt J. Lanier S.M. J. Biol. Chem. 1995; 270: 15269-15276Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 4Sato M. Ribas C. Hildebrandt J.D. Lanier S.M. J. Biol. Chem. 1996; 271: 30052-30060Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). This approach resulted in the partial purification and characterization of the NG10815 G-protein activator that directly increased GTPγS 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-protein-coupled receptor (5Cismowski M. Takesono A. Ma C. Lizano J.S. Xie S. Fuernkranz H. Lanier S.M. Duzic E. Nature Biotechnol. 1999; 17: 878-883Crossref PubMed Scopus (158) Google Scholar). The pheromone response pathway in S. cerevisiaeincorporates a seven-membrane span receptor, a heterotrimeric G-protein, and a mitogen-activated protein kinase cascade that regulates mating behavior and growth (6Schultz J. Ferguson B. Sprague Jr., G.F. Curr. Opin. Genet. Dev. 1995; 5: 31-37Crossref PubMed Scopus (48) Google Scholar). In this report, we present the identification and characterization of two proteins isolated in this functional screen. These proteins were termed AGS21 and AGS3 foractivators of G-proteinsignaling. The NG108-15 cell line was propagated as described previously (4Sato M. Ribas C. Hildebrandt J.D. Lanier S.M. J. Biol. Chem. 1996; 271: 30052-30060Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). mRNA was prepared from twenty 100-mm confluent plates of cells and was used to generate a cDNA library in the yeast expression vector pYES2 by standard procedures using reagents from Stratagene. The NG108-15 cDNA library was screened for activators of the pheromone response pathway as described (5Cismowski M. Takesono A. Ma C. Lizano J.S. Xie S. Fuernkranz H. Lanier S.M. Duzic E. Nature Biotechnol. 1999; 17: 878-883Crossref PubMed Scopus (158) Google Scholar). We used a his3 far1 yeast strain containing a genomic integration of the FUS1p-HIS3 reporter construct and lacking both the native pheromone response receptor Ste3 and Gpa1 Gα subunit. A plasmid carrying a modified mammalian Giα2 subunit in which the amino terminus was replaced with the first 41 amino acids of Gpa1 was introduced into this strain. Essentially, the screen took advantage of the inducible expression of the library cDNAs by differential plating of transformants on selective medium. A rat brain cDNA library was screened with a 32P-labeled 43-mer oligonucleotide (5′-ATAAGGCTGAAGAAATCCTCATCAGGCATGGTAGGGCCAC-3′) derived from the AGS3 sequence cDNA isolated in the yeast screen. The longest brain AGS3 cDNA still contained a truncated reading frame, and the 5′-end was extended by 5′-RACE using Marathon-Ready cDNA (CLONTECH) from rat brain. Domain searches were performed by the GCG COMPARE/DOTPLOT PROFILESEARCH and MOTIFS commands via internet access to the simple modular architecture research tool (SMART) data bases (7Schultz J. Milpetz F. Bork P. Ponting C.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5857-5864Crossref PubMed Scopus (2992) Google Scholar). Multiple expectation maximization for motif elucidation version 2.2 (MEME), motif alignment and search tool (MAST), and profile searches were performed via web resources at the San Diego SuperComputer Center (8Bailey T.L. Gribskov M.J. J. Comput. Biol. 1997; 4: 45-59Crossref PubMed Scopus (58) Google Scholar). Secondary structure analysis was performed with the GCG PEPPLOT, HELICALWHEEL, and MOMENT programs as well as by the PROBE program, the UCLA/DOE Fold Prediction Server, and the PredictProtein network server at EMBL-Heidelberg (9Fischer D. Eisenberg D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11929-11934Crossref PubMed Scopus (104) Google Scholar, 10Holbrook S.R. Dubchak I. Kim S.H. BioTechniques. 1993; 14: 984-989PubMed Google Scholar). Protein interaction assays with purified G-protein subunits, GST fusion proteins, and immunoblotting were conducted as described previously (2Wu G. Hildebrandt J. Benovic J.L. Lanier S.M. J. Biol. Chem. 1998; 273: 7197-7200Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). For protein interaction studies in crude cell lysates, confluent 100-mm dishes of DDT1-MF2 cells were lysed in 50 mmTris-HCl, pH 7.4, 5 mm EDTA, 150 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 60 μmbenzamidine, 40 μm pepstatin, 1% Nonidet P-40 (500 μl/dish) and were centrifuged at 14,000 × g. 225 μl of lysate containing GDP (30 μm) or GTPγS (30 μm) were incubated with GST or GST·AGS3 (300 nm) for 30 min at 24 °C or at 4 °C for 12 h. 25 μl of a 50% slurry of glutathione-Sepharose were added, and the incubation continued for 1 h. Samples were then centrifuged, and the matrix was washed with 2 × 300 μl of lysis buffer prior to solubilization and denaturing gel electrophoresis (2Wu G. Hildebrandt J. Benovic J.L. Lanier S.M. J. Biol. Chem. 1998; 273: 7197-7200Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). For immunoprecipitation, antiserum generated against the carboxyl terminus of Gi3 (1 to 50 dilution of sera) was incubated with 250 μl of precleared lysate for 16 h at 4 °C, and immune complexes were isolated on Gammabind G-Sepharose. Bound proteins were detected by immunoblotting membrane transfers with specific antisera. The peptide C-VDLAGSPEQEASGLPDPQQQYPPGAS was used for generation of AGS3 antisera in rabbits. To facilitate the identification of novel receptor-independent activators of heterotrimeric G-proteins, we developed an expression cloning system based upon the pheromone response pathway in S. cerevisiae. We used a yeast strain that lacked the pheromone receptor and contained a modified mammalian G-protein (Giα2) in place of the yeast G-protein Gpa1 (5Cismowski M. Takesono A. Ma C. Lizano J.S. Xie S. Fuernkranz H. Lanier S.M. Duzic E. Nature Biotechnol. 1999; 17: 878-883Crossref PubMed Scopus (158) Google Scholar, 11Klein C. Paul J.I. Sauve K. Schmidt M.M. Arcangeli L. Ransom J. Trueheart J. Manfredi J.P. Broach J.R. Murphy A.J. Nature Biotechnol. 1998; 16: 1334-1337Crossref PubMed Scopus (130) Google Scholar). The yeast strain was further modified to respond to activation of the pheromone response pathway with a readout of growth (5Cismowski M. Takesono A. Ma C. Lizano J.S. Xie S. Fuernkranz H. Lanier S.M. Duzic E. Nature Biotechnol. 1999; 17: 878-883Crossref PubMed Scopus (158) Google Scholar). As an initial source of such G-protein activators (4Sato M. Ribas C. Hildebrandt J.D. Lanier S.M. J. Biol. Chem. 1996; 271: 30052-30060Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) we generated a NG108-15 cDNA library in the galactose inducible yeast expression vector pYES2. Three rounds of transformation/screening with different libraries yielded three distinct cDNAs that promoted growth in a galactose-dependent manner (Fig.1, A and B). Epistasis experiments indicated that cDNA #34, which was the weakest of the three in the yeast screen, acted downstream of Ste5 (a component of the yeast mitogen-activated protein kinase cascade) in the pheromone response pathway, and this protein will be described elsewhere. cDNAs #37 and 53 did not function in the null Ste5 genetic background and were also inactive in yeast strains lacking Ste20 (yeast homolog of p21-activated kinases) or Ste4 (yeast homolog of Gβ), indicating that these proteins activated the pheromone response pathway at or near the level of G-protein (Fig.1 B). Efforts were focused on cDNAs #37 and 53. Immunoblot analysis indicated that neither cDNA altered the levels of Gα or Gβ subunits (data not shown). The selectivity of the two cDNAs for different G-protein heterotrimers was determined using yeast strains expressing Gpa1 (yeast Gα), Gsα, Giα2(Gpa1(1–41)), Giα3(Gpa1(1–41)), and Gα16(Gpa1(1–41)). In β-galactosidase reporter assays, cDNA #37 was active in each of the genetic backgrounds except Gpa1, whereas cDNA #53 was only active in the Giα2 and Giα3genetic backgrounds (Fig.2 A). The preceding observations indicated that the activation of the pheromone response pathway by cDNAs #37 and 53 depended upon the presence of heterotrimeric G-proteins and the composition of subunit isoforms. cDNAs isolated via this expression cloning system were therefore named activators of G-protein signaling (AGS). cDNAs #37 and 53 were termed AGS2 and AGS3, respectively. AGS1, isolated from a human liver cDNA library, encodes a novel Ras-related protein (5Cismowski M. Takesono A. Ma C. Lizano J.S. Xie S. Fuernkranz H. Lanier S.M. Duzic E. Nature Biotechnol. 1999; 17: 878-883Crossref PubMed Scopus (158) Google Scholar). 2M. Cismowski, C. Ma, C. Ribas, X. Xie, M. Spruyt, J. S. Lizano, S. M. Lanier, and E. Duzic, submitted for publication. Figure 2Functional and biochemical properties of AGS2 and AGS3. A, bioactivity of cDNA #37 and 53 in yeast strains expressing different Gα subunits. The Gpa1, Giα2(Gpa1(1–41)), Giα3(Gpa1(1–41)), and Gα16(Gpa1(1–41)) yeast strains expressed similar amounts of G-protein as determined by immunoblotting with Gpa1-specific antisera. Each series of experiments include data obtained in the absence of Gα to indicate the signal obtained when the action of Gβγ is not limited by reformation of heterotrimer. Data are presented as the mean ± S.E. of three experiments with duplicate determinations. B, interaction of AGS2 and AGS3 with G-proteins. AGS2 and the bioactive AGS3 peptide (74 amino acids) beginning at the first in frame methionine were generated as GST fusion proteins. GST fusion proteins were incubated with G-protein subunits (Gα = 60–80 nm, Gβγ = 40 nm, fusion protein = ∼300 nm, total volume = 250 μl) for 12 h at 4 °C. GDP = 10 μm. GTPγS = 10 μm plus 5 mmMgCl2. Proteins were then adsorbed to glutathione matrix and retained G-protein subunits identified by immunoblotting following gel electrophoresis. Giα2 was generated in Sf9 cells as an amino-terminal His6-tagged protein and was detected using anti-Xpress antisera (Invitrogen). Input, 20 μl of the incubation mixture. The results presented are representative of four separate experiments. The GST·AGS3 fusion protein was functionally similar to the original AGS3 isolate in terms of its ability to promote growth. In contrast, the GST·AGS2 fusion protein did not promote growth in the yeast assay system, suggesting that the amino-terminal region of AGS2 has an important functional role. C, effect of G204A Giα2substitution and RGS4 on the functionality of AGS proteins. AGS1 was discovered in a similar screen using a human liver cDNA library (5Cismowski M. Takesono A. Ma C. Lizano J.S. Xie S. Fuernkranz H. Lanier S.M. Duzic E. Nature Biotechnol. 1999; 17: 878-883Crossref PubMed Scopus (158) Google Scholar). WT, wild type strain CY1316/1183 containing Giα2 as described for the original yeast screen. Similar results were obtained in three experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) AGS2 (770 nt) encoded a protein identical to mouse Tctex1, a light chain component of the cytoplasmic motor protein dynein and the flagellar dynein inner arm (12King S.M. Dillman III, J.F. Benashski S.E. Lye R.J. Patel-King R.S. Pfister K.K. J. Biol. Chem. 1996; 271: 32281-32287Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 13Harrison A. Olds-Clarke P. King S.M. J. Biol. 1998; PubMed Scopus Google Scholar). also in the cell of dynein may subserve as in signaling J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). with the G-protein-coupled receptor the kinase and a putative of F. S. A. G. M. E. J. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus (58) Google Scholar, S. M. S. A. J. 1998; Google Scholar, C. U. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The functionality of AGS2 in the yeast assay system also a direct interaction with heterotrimeric G-proteins. This was in protein interaction studies using a GST·AGS2 fusion protein and purified G-protein subunits. AGS2 did not with Giα2 directly bound brain Gβγ (Fig.2 B). The bioactivity associated with AGS2 in the yeast assay system a regulatory of heterotrimeric G-protein subunits in dynein both Gα and Gβγ are in of membrane and A. J. C. J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, S. M.M. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar, J. Biol. 1996; PubMed Scopus Google Scholar, C. J. J. J.R. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). AGS3 of and contained a truncated reading frame at its terminus with a methionine in a sequence for The of this reading frame (74 amino acids) was by analysis and immunoblotting with specific peptide antisera (data not shown). The AGS3 peptide was generated as a GST fusion protein and for specific with purified G-protein subunits 2 B). AGS3 bound did not with purified brain Gβγ 2 B). AGS3 the of Gα and exhibited selectivity for Gα subunits as in the functional assays described B). The protein interaction data and the functional data in yeast indicated that AGS2 and AGS3 activated the pheromone response pathway by a that heterotrimeric G-protein. As an initial approach to define of action in the context of the we the yeast We yeast strains in which signal processing in the pheromone response pathway was by of the with a G204A Giα2 or of the protein RGS4 1996; PubMed Scopus Google Scholar). as a Gα subunit by of its for and is of signal by an activated receptor in our yeast J. Cismowski, A. S. M. Lanier, and E. Duzic, AGS2 and AGS3 were G-proteins by a similar to a G-protein-coupled receptor, then action be in such genetic this was not the (Fig.2 In contrast, AGS protein isolated in the functional screen and described (5Cismowski M. Takesono A. Ma C. Lizano J.S. Xie S. Fuernkranz H. Lanier S.M. Duzic E. Nature Biotechnol. 1999; 17: 878-883Crossref PubMed Scopus (158) Google was not active in the genetic backgrounds as a AGS2 and AGS3 also did not GTPγS binding to heterotrimeric brain G-protein or Giα2 (data not which with the of AGS1 and the NG108-15 G-protein activator (4Sato M. Ribas C. Hildebrandt J.D. Lanier S.M. J. Biol. Chem. 1996; 271: 30052-30060Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, M. Takesono A. Ma C. Lizano J.S. Xie S. Fuernkranz H. Lanier S.M. Duzic E. Nature Biotechnol. 1999; 17: 878-883Crossref PubMed Scopus (158) Google These data suggest that AGS2 and AGS3 the pheromone response pathway at the level of G-protein, this not require the generation of binding to G-protein subunits, AGS2 and AGS3 may heterotrimer formation or promote subunit mechanisms of G-protein activation from that involving a typical G-protein-coupled receptor and to selective activation of AGS2 and AGS3 may G-proteins within a signal transduction complex or regulate signal processing by that Gα and Gβγ may of each other in the and AGS2 and AGS3 serve as alternative functional binding partners for the G-protein subunits Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar, J. Biol. 1996; PubMed Scopus Google Scholar, C. J. J. J.R. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). A rat AGS3 cDNA amino was identified by a rat brain cDNA library and 5′-RACE A). The AGS3 generated as an amino-terminal protein in Sf9 also with GαGDP as described for the AGS3 isolated in the yeast screen. AGS3 and complexes within the cell as determined in experiments with crude cell and S. M. Lanier, analysis of and indicated that AGS3 exhibited with the partial mouse cDNA the human protein and a protein was isolated as a truncated in a screen using Giα2 as G. B. 1996; PubMed Scopus Google Scholar). of human and mouse data bases indicated that AGS3 and are not and likely two distinct of a protein by and functional AGS3, and the C. protein two that the protein in The amino-terminal of the AGS3 of which serve as protein interaction and regulatory in proteins. The in AGS3 may function as a regulatory the bioactivity of the region or the of AGS3 and associated proteins within the The of AGS3 and the two proteins four of amino acids that One or two such are also in four proteins that the properties or of G-proteins B). was isolated in a yeast screen using as and may as a J. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). is a protein for the G-protein and was also isolated in a screen using as J.D. R. J. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). and are of the of of G-protein The presence of such a motif and we to the sequence in 2 as a G-protein regulatory motif. The motif may be a for proteins that with G-proteins and regulate subunit and/or the interaction of the G-protein with other entities in signal analysis indicated that each motif as an The AGS3 cDNA isolated in the yeast screen contained one motif. of into this motif that the interaction of AGS3 with Giα2 and in crude cell and the fusion proteins were inactive in the yeast assay system (data not shown). As for the interaction AGS3 and purified Giα2 (Fig.2 AGS3 with GTPγS in the crude cell lysate the presence of which the G-protein the complex from the mammalian cell lysate did not In contrast, Gβγ subunits were detected when GαGDP was isolated from the cell by with a Gα subunit These data the that AGS3 G-protein signaling by subunit is that AGS3 is selectively with a of Gα in the cell that of Gβγ and unexpected functional AGS proteins are of a of accessory proteins that signal by heterotrimeric G-protein systems (3Sato M. Kataoka R. Dingus J. Wilcox M. Hildebrandt J. Lanier S.M. J. Biol. Chem. 1995; 270: 15269-15276Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 4Sato M. Ribas C. Hildebrandt J.D. Lanier S.M. J. Biol. Chem. 1996; 271: 30052-30060Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, S. M.M. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, J. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, J.D. R. J. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). entities may the of activated G-protein within the cell of or provide a for signal amplification by in with G-protein-coupled proteins also provide a for signal input to heterotrimeric G-protein signaling systems that is distinct from that initiated by a seven-membrane span hormone receptor. of distinct AGS2 and AGS3 may also to a of proteins that serve as binding partners for Gα and Gβγ the subunits to subserve functions that do not require initial heterotrimer formation. We and of for We for the to these studies and of and D. Hildebrandt of for of the and We D. Hildebrandt of for purified brain and of for