It is well established that exposure of target cells to parathyroid hormone (PTH) in vivo or in vitro decreases their subsequent adenylate cyclase (AC) response to PTH (desensitization). In cultured chick embryonic bone cells (CCEBC), prior exposure to PTH results in nearly complete desensitization with only partial loss of PTH receptors, as assessed by radioligand binding assays. The long-range goal of this proposal is to define the molecular basis for this """"""""uncoupling"""""""" of the PTH receptor in desensitized bone cells. Specifically, these studies are designed to test the hypothesis that PTH receptors in desensitized CCEBC are functionally and structurally altered so that they are unable to transmit stimulatory signals to AC. Initial experiments will determine the conditions (time, PTH concentration) required for optimal uncoupling of the PTH receptor. The initial objective is to achieve the greatest degree of desensitization possible with the minimum perturbation of PTH-receptor binding. The ability of these uncoupled receptors, relative to normally coupled receptors, to interact with the guanyl nucleotide-dependent regulatory protein (N) of adenylate cyclase will then be assessed in two ways. First, comparisons will be made of the regulation of control and uncoupled receptors by exogenous guanyl nucleotide. This will involve computer-assisted analysis of the kinetics of PTH-receptor binding in membranes from CCEBC in the presence and absence of guanyl nucleotides. Secondly, the structural interaction of PTH receptors and N in the membrane will be assessed via covalent labeling of each component, followed by solubilization and chromatography in non-denaturing systems. In addition, structural changes in the PTH receptor upon desensitization will be assessed by photoaffinity laleling of the receptor, followed by gel electrophoresis. Included in these latter studies is an evaluation of whether the phosphorylation state of the PTH receptor is altered during desensitization. Further support for the notion that changes in the structure or function of PTH receptors relate causally to desensitization will be obtained by examining the correlation between the reversal of such effects and resensitization of CCEBC. It is anticipated that these studies will ultimately provide a framework for understanding the altered skeletal response to PTH seen in pathologic states in vivo (e.g., vitamin D deficiency, corticosteroid excess).
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