Abstract
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.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF107723
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
Epistasis experiments indicated that cDNA #34, which was the weakest of the three in the yeast screen, acted downstream of Ste5 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 inactive in yeast strains lacking Ste20 or Ste4, indicating that these proteins activated the pheromone response pathway at or near the level of G-protein (Fig. 1B)
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF107723. 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.
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