REGULATION OF THE PTH/PTHRP RECEPTOR

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Description:(Taken directly from the application) The diverse endocrine actions of PTH and paracrine actions of PTHrP are determined by the local concentrations of ligands and the responsiveness of target cells. Cellular responsiveness is determined by the number of receptors at the cell surface, the ability of receptors to convert ligand binding to G protein(s) activation, and the distal biological responses to G protein(s). This proposal will focus on understanding the post-translational events that regulate the responsiveness of the PTH/PTHrP receptor. We have powerful tools for probing the molecular mechanisms involved in each of these processes. These include specific receptor antibodies, green-fluorescent protein-tagged receptors, and libraries of mutant receptors (phosphorylation-deficient, constitutively-active, activation-deficient and Gq-deficient), PTH analogs (AC-selective) and stable bone-derived cell lines with different patterns of differentiation and activation of bone-specific genes in response to continuous versus intermittent PTH treatment. In Aim I the phosphorylation sites will be mapped and the role of receptor phosphorylation and of G protein receptor kineses in internalization and desensitization will be determined. In Aim II, mutant receptors, which dissociate binding from activation, will be used to determine if binding and/or activation is/are required for phosphorylation, internalization and/or desensitization. In Aim III the green fluorescent protein-tagged PTH/PTHrP receptors will be used to characterize the cellular proteins that associate with the internalized PTH/PTHrP receptor vesicles and to examine the role of receptor phosphorylation in this process. The physiological relevance of internalization and desensitization will be examined in the bone-derived cell lines that have different patterns of biologic responses to continuous versus intermittent PTH treatment. We propose in Aim IV to interfere with the desensitization process in these cells and determine how this interference affects the osteoblast's responses to intermittent versus continuous administration of PTH.