The overall objective of this research proposal is to investigate and define several of the biochemical and molecular mechanisms which regulate the expression and function of the parathyroid hormone receptor (PTHR) in human osteoblastic cells. PTH is one of the most important hormones in the physiology, pathology and, increasingly, in the pharmacology of bone and mineral metabolism. The first specific aim examines the initial molecular target of PTH action, its receptor, focusing on the mechanisms of regulation of receptor expression and function by its homologous ligands PTH and PTH-related protein (PTHrP). The experiments proposed are made possible by the availability of several critical new reagents including antibodies to the human (h) PTHR, novel PTH affinity probes, cloned G protein receptor kinases (GRKs) and dominant negative mutants of these kinases. The second specific aim follows from our recent findings that overexpression of two of the GRKs, betaARK-1 and betaARK-2, causes marked enhancement of the binding of PTH to its receptor, and focuses on elucidation of the role of GRKs in PTHR expression . The third specific aim results from our discovery that incubation of human osteoblast-like SaOS-2 cells with human placental alkaline phosphatase causes the PTHR- mediated signal transduction pathway to distinguish between the agonist actions of PTH and PTHrP, and focuses on explaining the biochemical mechanism of this ligand-specific discrimination. Although it is widely recognized that homologous and heterologous regulation of G protein-coupled receptors is physiologically and pharmacologically important, tint he case of the PTHR little mechanistic detail is known because the essential tools were not previously available. Results of the proposed experiments will fill considerably this gap in knowledge and will have significance because they will enhance understanding of how the PTHR on human osteoblasts can respond differentially to intermittent versus persistent elevations of PTH of PTHrP. Transient elevations of PTH elicit an anabolic response while persistent elevations cause bone catabolism. Understanding the biochemical basis of these two functionally distinct responses will have clear pathophysiological and potential therapeutic implications for such important clinical disorders as osteoporosis, hyperparathyroidism, and the hypercalcemias of cancer.
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