Residues in the first extracellular loop of a G protein-coupled receptor play a role in signal transduction

Abstract

The Saccharomyces cerevisiae pheromone, a-factor (WHWLQLKPGQPMY), and Ste2p, its G protein-coupled receptor, were used as a model system to study ligandreceptor interaction. Cys-scanning mutagenesis on each residue of EL1, the first extracellular loop of Ste2p, was used to generate a library of 36 mutants with a single Cys residue substitution. Mutation of most residues of EL1 had only negligible effects on ligand affinity and biological activity of the mutant receptors. However, five mutants were identified that were either partially (L102C and T114C) or severely (N105C, S108C, and Y111C) compromised in signaling but retained binding affinities similar to those of wild-type receptor. Threedimensional modeling, secondary structure predictions, and subsequent circular dichroism studies on a syn. thetic peptide with amino acid sequence corresponding to EL1 suggested the presence of a helix corresponding to EL1 residues 106 to 114 followed by two short β-strands (residues 126 to 135). The distinctive periodicity of the five residues with a signal-deficient phenotype combined with biophysical studies suggested a functional involvement in receptor activation of a face on a 310 helix in this region of EL1. These studies indicate that EL1 plays an important role in the conformational switch that activates the Ste2p receptor to initiate the mating pheromone signal transduction pathway.