I propose to investigate the interaction of parathyroid hormone (PTH) and PTH-related peptide (PTHrP) with three G-protein-coupled receptors, the PTH/PTHrP receptor for (P1R), the newly discovered PTH-2 receptor (P2R), and the third PTH receptor (P3R) recently described in zebrafish. P1R and P2R bind PTH and PTHrP with equally high affinity. P2R binds PTH preferentially, while P3R binds preferentially, to PTHrP. The carboxy terminus of PTH/PTHrP is known to be important for high affinity binding to these receptors. Photoaffinity cross-linking studies will therefore be performed to define receptor regions that interact with the carboxy-terminus of PTH/PTHrP. Site-directed mutagenesis of the receptors will then be used to identify individual receptor residues that interact with each ligand. These studies will thus attempt to define the receptor domains and specific residues responsible for high affinity binding to PTH and PTHrP, helping to determine how P2R and P3R distinguish between the two ligands. The information obtained will aid in the development of nonpeptide agonists and/or antagonists that may gain clinical significance in treating osteoporosis or the various forms of hyperparathyroidism and hypoparathyroidism.