Peptide hormones influence the metabolism of virtually every eukaryotic cell by initiating signal transduction cascades following interaction with receptors on cell membranes. A multitude of human diseases are linked to hormone and receptor pathology making an understanding of how the hormone-receptor complex is formed and how the subsequent signal transduction is triggered essential for developing medicines to treat many diseases. We propose to continue our studies on the interaction between alpha-factor, a tridecapeptide Saccharomyces cerevisiae pheromone, and its receptor (Ste2p), a member of the ubiquitous seven transmembrane domain, G protein-coupled receptor family. In recent months we have made exciting progress in the purification and reconstitution of the receptor and in the development of photoactivatable alpha-factor analogs. These tools will be used to crosslink peptide analogs into the receptor to map the specific residues that participate in the binding of the alpha-factor. Concurrently, fluorescent analogs will be used to discern the molecular environment of the peptide-binding pocket, and molecular biological approaches will be used to generate mutant receptors to verify and expand upon the binding studies. Segments of the of Ste2p will be synthesized or biosynthesized and their structure will be studied in lipid-like environments using circular dichroism, high resolution solution NMR spectroscopy and solid-state NMR spectroscopy. Interactions among the transmembrane segments of the receptor will be studied by both biophysical and molecular biological methods. Fluorescently labeled receptors will be used in studies designed to determine the conformational changes in the receptor induced by ligand binding. Overall, these studies will provide the basis for building a testable model revealing the ligand binding site of the receptor, uncovering interactions among receptor transmembrane segments, and demonstrating changes in receptor structure at the molecular level on ligand activation. In general, these studies should provide insights into the function of peptide-activated G-protein-coupled receptors.
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