Exposure of cultured chick embryonic osteoblast-like cells (OB) to doses of parathyroid hormone (PTH) that cause essentially complete desensitization of PTH-stimulated cyclic AMP production decreases specific cell surface PTH binding by less than 50%. Thus, the loss of cell surface PTH receptors does not entirely account for the magnitude of desensitization observed, suggesting that a fraction of cell surface PTH receptors in desensitized OB are uncoupled from cyclic AMP production. Using monoclonal antibodies (MAbs) developed recently in our laboratory to immunoprecipitate the PTH receptor, we have obtained preliminary evidence that the PTH receptor in OB is phosphorylated and that exposure of OB to PTH rapidly (1 min) increases receptor phosphorylation to a maximum at a time (30 min) when cells are maximally desensitized. Our long-term objective is to test the hypothesis that the steady-state level of functional PTH receptors in the OB plasma membrane is regulated by their level of phosphorylation and that modulators of PTH effects might employ a phosphorylation- dephosphorylation mechanism to variably dampen or enhance PTH action, respectively.
The Specific Aims of the present proposal are designed to provide multiple lines of evidence that either support or reject this hypothesis. With few exceptions, we have had considerable experience with the principal technology needed to perform the work proposed in this application. We have selected the phenomenon of homologous desensitization as a framework to investigate the significance of PTH receptor phosphorylation under conditions in which OB receptors are unoccupied or occupied with PTH agonist or PTH antagonist. Furthermore, PTH receptor MAbs will be used to immunoprecipitate phosphorylated PTH receptors. These procedures should enhance our ability to interpret results by providing evidence of both biologic and immunologic specificity. Investigations in our and other laboratories should result in the cloning of the PTH receptor in the foreseeable future. It will thus be possible to perform studies of the influence of specific mutations of phosphorylation sites on PTH receptor function. Successful completion of the studies proposed in the present application will form the basis for the design of these molecular investigations and should help provide important biologic perspective for the interpretation of their results.
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