G protein-coupled receptors (GPCRs) comprise the largest family of eukaryotic membrane proteins. Despite intense efforts by many laboratories over the last twenty years, there remains much to be learned about how ligand binding initiates signal transduction and how GPCRs are regulated. The long-term goal of this research is to use the Saccharomyces cerevisiae pheromone alpha-factor and its receptor Ste2p to provide fundamental insights into the recognition of medium-sized peptide ligands by GPCRs, to develop approaches that can be used to acquire information on the structure of these membrane proteins in the resting and ligand-activated state, and to learn about the regulation of GPCR function. Specifically, we intend to use biochemical, molecular biological, and biophysical approaches to: (i) determine the energetics of the nteractions between the tridecapeptide alpha-factor and Ste2p; (ii) elucidate the structure of the ligandreceptor complex in the active and inactive state; (iii) develop approaches to solve the three-dimensional structure of domains of GPCRs and reveal conformational changes in the extracellular regions of Ste2p that are triggered by agonist binding; and (iv) identify regulatory proteins that interact with the carboxyl-terminus of Ste2p. To accomplish these aims we will use a variety to genetic, biochemical, and biophysical techniques including site-directed mutagenesis, unnatural amino acid replacement, circular dichroism, nuclear magnetic resonance, mass spectrometry, fluorescence resonance energy transfer, and peptide synthesis using solid state methods, native chemical ligation, and bacterial expression. Success in these studies should provide molecular level information about the structure, function, and regulation of GPCRs and further develop tools that can be used by investigators studying human GPCRs. Medical Relevance: Peptide hormones control many essential biological processes in all organisms and tissues. Mutations in genes encoding hormone receptors lead to many pathological conditions in humans, and GPCRs are the target of a majority of pharmacological agents used in medicine today. An understanding of the interaction of ligands with GPCRs and how these interactions trigger signal transduction and are regulated is necessary to facilitate the design and synthesis of drugs to treat many diseases.
Showing the most recent 10 out of 31 publications
