Yeast α Mating Factor Structure-Activity Relationship Derived from Genetically Selected Peptide Agonists and Antagonists of Ste2p

Abstract

␣-Factor, a 13-amino-acid pheromone secreted by haploid ␣ cells of Saccharomyces cerevisiae, binds to Ste2p, a seven-transmembrane, G-protein-coupled receptor present on haploid a cells, to activate a signal transduction pathway required for conjugation and mating. To determine the structural requirements for ␣-factor activity, we developed a genetic screen to identify from random and semirandom libraries novel peptides that function as agonists or antagonists of Ste2p. The selection scheme was based on autocrine strains constructed to secrete random peptides and respond by growth to those that were either agonists or antagonists of Ste2p. Analysis of a number of peptides obtained by this selection procedure indicates that Trp1, Trp3, Pro8, and Gly9 are important for agonist activity specifically. His2, Leu4, Leu6, Pro10, a hydrophobic residue 12, and an aromatic residue 13 are important for both agonist and antagonist activity. Our results also show that activation of Ste2p can be achieved with novel, unanticipated combinations of amino acids. Finally, the results suggest the utility of this selection scheme for identifying novel ligands for mammalian G-protein-coupled receptors heterologously expressed in S. cerevisiae.Haploid Saccharomyces cells of mating type ␣ secrete a 13-amino-acid pheromone, ␣-factor (Trp1-His2-Trp3-Leu4-Gln5-Leu6-Lys7-Pro8-Gly9-Gln10-Pro11-Met12-Tyr13), that can bind and stimulate Ste2p, a G-protein-coupled receptor expressed on the surface of haploid cells of mating type a (reviewed in reference 26). Binding of ␣-factor to Ste2p activates the pheromone signaling pathway by promoting on the cytoplasmic side of the plasma membrane dissociation of a coupled heterotrimeric G protein into its constituent ␣ subunit and ␤␥ complex. The ␤␥ complex in conjunction with Cdc42p initiates a kinase cascade that results in activation of mitogenactivated protein kinase homologs, Kss1p and Fus3p (reviewed in reference 17). These activated kinases promote both cell cycle arrest, through inactivation of the cyclin B/p34 cdc28 complex in a process mediated by Far1p, and transcription of a variety of genes required for mating and conjugation, mediated by the Ste12p transcriptional activator. Activation of this signal transduction pathway is essential for productive mating, and mutations that attenuate this pathway result in sterility.Current knowledge of ␣-factor suggests a complex structurefunction relationship. Although determinants of its various biological activities are not restricted to discrete peptide regions, the determinants may be more heavily concentrated in certain domains. For example, residues that initiate signaling may be concentrated in the N terminus, while those that mediate binding may dominate the C-terminal region. This suggestion is based on observations that removal or substitution of Trp1, His2, or Trp3 results in loss of biological activity that exceeds the relatively small reductions in binding affinity (10,22). For example, removal of Trp1 to form des-Trp1-␣-factor yields a pep...

A G Protein-coupled Receptor for UDP-glucose

“…Activation of KIAA0001 Expressed in Yeast Cells-As part of a large program to identify the natural ligands for orphan GPCRs, we expressed a number of known and orphan GPCRs, including KIAA0001, in yeast strains engineered to respond to agonist activation with increased expression of a pheromone signaling pathway-inducible FUS1-lacZ reporter gene (7,8); expression of this gene is easily monitored by a simple colorimetric assay of enzyme activity. As one cannot predict the specific G protein that will transduce signals from a given orphan receptor, we expressed receptors in several yeast FIG.…”

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Identification of Allosteric Peptide Agonists of CXCR4

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Long-chain alkyl ester of AMP acts as an antagonist of glucose-induced signal transduction that mediates activation of plasma membrane proton pump in Saccharomyces cerevisiae

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