(Taken directly from the application) The critical role that parathyroid hormone (PTH) plays in the control of blood mineral ion levels, and the importance of PTH-related peptide (PTHrP) in skeletal development and hypercalcemia of malignancy, emphasize the need to understand the molecular mechanisms by which these ligands bind to and activate their receptors. Our prior studies suggest that the ligands binding domain """"""""docks"""""""" to the N-terminal domain of the receptor, while the ligands N-terminal activation domain engages the receptor's """"""""core"""""""" region. But the specifics are vague. The goals of this proposal are to define the molecular details of the ligand-receptor interface. We will approach the problem by generating and analyzing altered ligands and mutant receptors. Ligand residues will be analyzed in the context of small fragments corresponding to the activation domain, PTH(1-14), and the binding domain, PTH(17-31). This will permit scanning and saturation substitution approaches involving multiple peptides. For key substitutions, corresponding """"""""intact"""""""" 1-34 or 1-31 peptides will be prepared for more extensive evaluations. Optimized activation and binding domains will be combined in """"""""minimized"""""""" model peptides. The ligand work will be guided by NMR structural analyses, to be performed by our collaborators Drs. Weiss and Hua. In parallel to the ligand studies, we will define ligand-binding sites in the receptor using receptor mutagenesis. Second-site suppression analysis, where mutant receptors will be tested for intermolecular rescue of specifically modified PTH analogs, will be used to define point-to-point interactions. In this regard, we will define the molecular basis for ligand selectivity in the PTH-2 receptor, using mutant receptors and ligands modified at residues 5 and 23. To complement our functional studies, we will employ benzophenone (BPA)-containing ligands to cross-link ligand-receptor complexes. The topology and functional contribution of the seven helical domains of the receptor will be investigated using intramolecular second-site suppression analysis, and histidine-scanning strategies aimed to confer antagonist or agonist responsiveness to metal ions. As a longer-term goal, we explore the development of minimized soluble receptor fragments. The overall studies should provide important new information on the molecular determinants of ligand recognition and the mechanisms of ligand-induced receptor activation in PTH receptor systems.
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