This closely integrated research proposal is a broad-based effort to analyze fundamental aspects of the biology of parathyroid hormone. A four-fold research effort is proposed: Project I, Evolution of the PTH/PTHrP Receptor and Its Ligands, will determine the structures of PTH, PTHrP, and the PTH/PTHrP receptor in non-mammalian species. The characterization of these structures will provide crucial data for understanding structure-function relationships. The non-mammalian systems will also provide novel model systems for clarifying the functions of these signalling molecules; for example, the roles of PTHrP and PTHrP receptors in early Xenopus development will be studied. Project II, Structural Requirements for Interaction of PTH With Its Receptor, will analyze the functional correlates of the structure of the PTH molecule. Site-directed mutagenesis and chemical synthesis of PTH fragments and analogs will be used as complementary strategies for designing probes for use in these functional studies. Systematic mutagenesis of the cloned PTH/PTHrP receptor cDNA will make possible a detailed analysis of the contacts between PTH and its receptor and the mechanisms whereby binding of ligand on one side of the plasma membrane leads to a conformational change on the cytoplasmic surface. Project III, PTH/PTHrP Receptors: Structure, Signalling, and Regulation, will focus on the way the PTH/PTHrP receptor controls intracellular signalling. A systematic search for and analysis of alternative forms of the PTH/PTHrP receptor will define the relevant signalling molecules. Extensive mutagenesis will be performed to clarify which portions of the receptor activate G proteins; the molecular nature of these G proteins will be clarified. The structural determinants of receptor desensitization and down-regulation will be determined. Project IV, Second Messengers and Regulation of Cellular Function by PTH, will analyze the mechanisms whereby PTH modulates specific cellular activities. An attempt will be made to determine which second messenger pathways control a variety of biologic activities, such as cell proliferation and ion transport. These studies will exploit PTH analogs with novel signalling properties, mutant PTH receptors with altered signalling properties, and mutant cell lines with specific blocks in second messenger pathways, in order to understand the regulation of distal effects of PTH. These studies will provide a firm base for an understanding of diseases of the parathyroid gland and lay the groundwork for potential therapeutic agents for metabolic bone disease.
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