Probing the Binding Site of a Heptahelical Peptide Pheromone Receptor Using Photoaffinity Labelling, Site-Directed Mutagenesis and Spectroscopic Approaches

G protein-coupled receptors (GPCRs) are multifunctional proteins involved in cell-cell communication and represent the largest gene family in eukaryotes. Signal molecules recognized by these receptors include odorants, biogenic amines, peptides and proteins, and are involved in regulation of a plethora of biological processes including pain sensation, growth, blood pressure regulation and intermediary metabolism. Despite the importance of GPCRs, few details are available on the molecular aspects of ligand recognition or signal transduction. Ongoing studies in our laboratory use mating in Saccharomyces cerevisiae as a paradigm to understand various aspects of GPCR function. One specific goal of our work is to understand the nature of the interaction of the pheromone α-factor (WHWLQLKPGQPMY) with its GPCR (Ste2p). In this report, we discuss a multi-pronged approach utilizing fluorescence spectroscopy, site-directed mutagenesis, and photoaffinity labeling to define the interaction of this peptide pheromone with Ste2p.