Interaction between transmembrane domains five and six of the alpha-factorreceptor

The a-factor pheromone receptor (STE2) activates a G protein signal pathway that induces conjugation of the yeast Saccharomyces cerevisiae, Previous s tudies implicated the third intracellular loop of this receptor in G; prote in activation. Therefore, the roles of transmembrane domains five and six ( TMD5 and -6) that bracket the third intracellular loop were analyzed by sca nning mutagenesis in which each residue was substituted with cysteine. Out of 42 mutants examined, four constitutive mutants and two strong loss-of-fu nction mutants were identified, Double mutants combining Cys substitutions in TMD5 and TMD6 gave a broader range of phenotypes, Interestingly, a V223C mutation in TMD5 caused constitutive activity when combined with the L247C , L248C, or S251C mutations in TMD6, Also, the L226C mutation in TMD5 cause d constitutive activity when combined with either the M250C or S251C mutati ons in TMD6. The residues affected by these mutations are predicted to fall on one side of their respective helices, suggesting that they may interact . In support of this, cysteines substituted at position 223 in TMD5 and pos ition 247 in TMD6 formed a disulfide bond, providing the first direct evide nce of an interaction between these transmembrane domains in the ct-factor receptor. Altogether, these results identify an important region of interac tion between conserved hydrophobic regions at the base of TMD5 and TMD6 tha t is required for the proper regulation of receptor signaling.