This broad based tightly integrated research proposal is aimed at understanding the molecular, cellular and physiological actions of parathyroid hormone (PTH) on bone and kidney and mineral ion homeostasis. A long range goal is to understand the cellular basis of PTH actions which underlie its therapeutic value in osteoporosis. Project I. PTH and PTHrP Interaction with PTH Receptors. Study of the molecular structure of the active biomolecular complex of PTH and its classic receptor (PTHR1) will be pursued further by highly effective, powerful iterative testing approaches using multiple methods of molecular biology and peptide chemistry with structurally minimized ligands and multiple receptor mutants. Project II. Regulation of the PTH/PTHrP Receptor. The biochemical and physiological consequences of altering the normally occurring modulation of surface expression of PTHR1 on target cells will be analyzed in vitro and in vivo with receptor constructs mutagenized to prevent phosphorylation and impair desensitization and internalization of the receptor. Project III. Second Messengers in PTH Action. Evidence that induced selectivity among the diverse signalling pathways of PTHR1 stimulated by PTH lead to distinctive biological responses in bone will be explored by administration of signal-selective PTH ligands in vivo. Cellular mechanisms responsible for altered target cell response will be addressed in vitro with bone-cell lines containing signal selective variants of the PTHR1. Project IV. Role of PLC in PTH Signalling: Mutant Receptors in Vivo. Evidence that abrogation of PLC- dependent PTHR1 signalling leads to abnormalities in bone development, & post-natal bone structural integrity and inducible abnormalities in mineral ion homeostasis (especially through renal mechanisms) will be explored by extensive studies in vivo with a genetically modified mouse strain that lacks signaling through the PLC pathway and mechanism-based studies with cells derived from the mutant mice in vitro. Project V. NHERF- Mediated PTH Signalling in Mineral Ion Homeostasis. NHERF proteins function as part of intracellular scaffold protein complexes that modulate cellular signaling. NHERFs have recently been shown to act as """"""""molecular switches"""""""" that increase activation by PTH of PLC-dependent pathways while suppressing adenyl cyclase pathways. The skeletal role of the NHERF proteins will be studied in vivo (using NHERF-1 knockout animals recently reported to have osteopenia) and in vitro with renal and bone cell lines, the latter derived from knockout animals and/or transfected with NHERF proteins. A versatile peptide core for supply of vital reagents and a newly established Skeletal Phenotyping Core with novel tools for analyses of induced skeletal responses provide critical support for the five projects.
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