Aromatic residues at the extracellular ends of transmembrane domains 5 and 6 promote ligand activation of the G protein-coupled α-factor receptor

The α-factor receptor (STE2) stimulates a G protein signaling pathway that promotes mating of the yeast Saccharomyces cerevisiae. Previous random mutagenesis studies implicated residues in the regions near the extracellular ends of the transmembrane domains in ligand activation. In this study, systematic Cys scanning mutagenesis across the ends of transmembrane domains 5 and 6 identified two residues, Phe²⁰⁴ and Tyr²⁶⁶, that were important for receptor signaling. These residues play a specific role in responding to α-factor since the F204C and Y266C substituted receptors responded to an alternative agonist (novobiocin). To better define the structure of this region, the Cys-substituted mutant receptors were assayed for reactivity with a thiol-specific probe that does not react with membrane-imbedded residues. A drop in reactivity coincided with residues likely to be buried in the membrane. Interestingly, both Phe²⁰⁴ and Tyr²⁶⁶ are located very near the interface region. However, these assays predict that Phe²⁰⁴ is accessible at the surface of the receptor, consistent with the strong defect in binding α-factor caused by mutating this residue. In contrast, Tyr²⁶⁶ was not accessible. This correlates with the ability of Y266C mutant receptors to bind α-factor and suggests that this residue is involved in the subsequent triggering of receptor activation. These results highlight the role of aromatic residues near the ends of the transmembrane segments in the α-factor receptor, and suggest that similar aromatic residues may play an important role in other G protein-coupled receptors.