IDENTIFICATION OF A POLAR-REGION IN TRANSMEMBRANE DOMAIN-6 THAT REGULATES THE FUNCTION OF THE G-PROTEIN-COUPLED ALPHA-FACTOR RECEPTOR

The alpha-factor pheromone receptor (Ste2p) of the yeast Saccharomyces cerevisiae belongs to the family of G protein-coupled receptors that contain seven transmembrane domains (TMDs), Because polar residues can influence receptor structure by forming intramolecular contacts betwe en TMDs, we tested the role of the five polar amino acids in TMD6 of t he alpha-factor receptor by mutating these residues to nonpolar leucin e, Interestingly, a subset of these mutants showed increased affinity for ligand and constitutive receptor activity, The mutation of the mos t polar residue, Q253L, resulted in 25-fold increased affinity and a 5 -fold-higher basal level of signaling that was equal to about 19% of t he alpha-factor induced maximum signal. Mutation of the adjacent resid ue, S254L, caused weaker constitutive activity and a 5-fold increase i n affinity. Comparison of nine different mutations affecting Ser(254) showed that an S254F mutation caused higher constitutive activity, sug gesting that a large hydrophobic amino acid residue at position 254 al ters transmembrane helix packing, Thus, these studies indicate that Gl n(253) and Se-254 are likely to be involved in intramolecular interact ions with other TMDs, Furthermore, Gln(253) and Ser(254) fall on one s ide of the transmembrane helix that is on the opposite side from resid ues that do not cause constitutive activity when mutated. These result s suggest that Gln253 and Ser(254) face inward toward the other TMDs a nd thus provide the first experimental evidence to suggest the orienta tion of a TMD in this receptor. Consistent with this, we identified tw o residues in TMD7 (Ser(288) and Ser(292)) that are potential contact residues for Gln(253) because mutations affecting these residues also cause constitutive activity, Altogether, these results identify a new domain of the alpha-factor receptor that regulates its ability to ente r the activated conformation.