The alphaFactor receptor is the first component of a signal transduction pathway that leads to cellular differentiation and mitotic growth arrest in Saccharomyces cerevisiae. This receptor is a member of a super-family of 7-transmembrane receptors involved in such diverse physiological processes as endocrine signaling, neurotransmission, vision, and blood- clotting. The function of these receptors is to sense low abundance signals impinging from outside the cell and to relay that signal to a tightly associated G-protein. Disruption of these G-protein coupled receptor complexes by mutation is known to cause a number of human diseases such as neonatal severe hyperparathyroidism, precocious puberty, retinitis pigmentosa, and various neoplasms. The yeast pheromone-response pathway is activated when a secreted 13-amino acid residue peptide (alphaFactor) binds its cognate receptor. The mechanism by which the alphaFactor receptor recognizes this peptide ligand and activates the internally-located G-alpha subunit is not known. The purpose of this investigation is to define the peptide binding site on the receptor and to study the physical properties of the receptor-ligand complex in both activated and unactivated states. The alphaFactor receptor will be overexpressed in yeast with an epitope-His6 tag appended to the C- terminus. Mapping of the peptide binding site will be done with a combination of N-bromoacetyl- and benzoylphenylalanine-alphaFactor peptide affinity labels. N-terminally truncated alphaFactor peptide affinity labels are antagonists and will serve as probes of the unactivated peptide-receptor complex. The labeled-receptor-His6 protein will be retrieved from crude membrane preparations by Ni-chelation chromatography and preparative polyacrylamide gel electrophoresis using techniques established by our group. Localization of the site of labeling will be done by epitope mapping, electrospray mass spectrometry, and sequencing of proteolytic fragments. Site-specific mutagenesis of the receptor will confirm these mapping studies. The receptor will be purified from yeast membranes by peptide affinity columns and by affinity tags incorporated into the receptor itself. Biophysical analysis of receptor-ligand macromolecular complexes will define the conformational change that occurs upon ligand activation. These studies include determination of the orientation and solvent accessibility of bound peptide-fluorophores, and detection of the receptor conformational change in real time using fluorescent donor-acceptor tags on both peptide and the third intracellular loop of the receptor. Capillary electrophoresis will serve as a highly sensitive means to quantitate peptide-receptor binding in solution and to detect gross conformational changes upon ligand activation. These studies should provide the basis for understanding how this receptor activates the downstream signaling pathway and will serve as a general model for other G protein/receptor mutant-related human diseases.
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