Analysis of random PCR‐originated mutants of the yeast Ste2 and Ste3 receptors

The G protein‐coupled receptors Ste2 and Ste3 bind α‐ and a ‐factor, respectively, in Saccharomyces cerevisiae . These receptors share a similar conformation, with seven transmembrane segments, three intracellular loops, a C‐terminus tail, and three extracellular loops. However, the amino acid sequences of these two receptors bear no resemblance to each other. Coincidently the two ligands, α‐ and a ‐factor, have different sequences. Both receptors activate the same G protein. To identify amino acid residues that are important for signal transduction, the STE 2 and STE 3 genes were mutagenized by a random PCR ‐based method. Mutant receptors were analyzed in MAT α cells mutated in the ITC 1 gene, whose product represses transcription of a ‐specific genes in MAT α . Expression of STE 2 or STE 3 in these cells results in autocrine activation of the mating pathway, since this strain produces the Ste2 receptor in addition to its specific ligand, α‐factor. It also produces a ‐factor in addition to its specific receptor, Ste3. Therefore, this strain provides a convenient model to analyze mutants of both receptors in the same background. Many hyperactive mutations were found in STE 3 , whereas none was detected in STE 2 . This result is consistent with the different strategies that the two genes have adopted to be expressed.