Parathyroid hormone (PTH) regulates calcium and phosphate homeostasis via direct, coordinated actions on bone and kidney. Most, if not all, of these effects are initiated by PTH receptor-mediated activation of the adenylate cyclase-cyclic AMP cascade. Despite the primary and necessary role of the PTH receptor in the hormone's effects, virtually nothing is known concerning the molecular basis of the receptor's ability to bind PTH (""""""""recognition"""""""") or to initiate biologic action (""""""""transduction""""""""). The present proposal constitutes a systematic approach to this problem. The specific objectives are: 1) To solubilize and partially purify the PTH receptor from bone and kidney in a functionally active state. The presence of receptor during the course of purification will be monitored through binding of 125I-PTH(1-34) either directly or after reconstitution of the receptor into liposomes and human red cell membranes, which lack PTH receptors. Retention of functional activity of the solubilized receptor will be demonstrated by its ability to reconstitute PTH-sensitive adenylate cyclase in cyc- S-49 lymphoma cell membranes that are deficient in the stimulatory guanine nucleotide regulatory protein (Ns). Purification will be effected using a combination of gel filtration and affinity chromatography, the latter employing immobilized PTH(1-34) as well as anti-receptor monoclonal antibodies. 2) To identify functional domains of the partially purified PTH receptor. In these studies, monoclonal antibodies specific for the """"""""recognition"""""""" and """"""""transduction"""""""" regions of the PTH receptor will be developed and employed as molecular probes of receptor structure. 3) To use monoclonal antibodies against the """"""""transducer"""""""" region of the PTH receptor to determine whether other adenylate cyclase-coupled hormone receptors have a homologous transduction domain. 4) To determine whether Ns from patients with pseudohypoparathyroidism is selectively defective in its ability to reconstitute high-affinity PTH receptors in erythrocytes, and PTH-responsive adenylate cyclase in S-49 cyc- membranes. Successful completion of these experiments would delineate the structural properties of PTH receptors required for hormone binding and signal generation, as well as the molecular basis for the genetic defect in PTH receptor-adenylate cyclase coupling present in pseudohypoparathyroidism. The anti-PTH receptor monoclonal antibodies will be useful for isolating pure populations of PTH-responsive bone and kidney cells, and, ultimately, for isolating genes encoding the PTH receptor.
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