Vasopressin is a hormone synthesized in the supraoptic and paraventricular nuclei of the hypothalmus and transported along axonal processes to the posterior pituitary where storage and subsequent secretion occur. Secretion of vasopressin occurs in response to osmotic (plasma osmolality) and nonosmotic (hypotension, hypovolemia) stimuli. Three types of vasopressin receptors have been identified based on functional assays and binding studies in the presence of agonists and antagonists. Vasopressin V1a receptors are located on hepatocytes and vascular smooth muscle cells while V1b receptors are located on cells in the adenohypophyseal region of the brain. These V1 receptors are coupled to phosphoinositol hydrolysis and increases in intracellular calcium. Vasopressin V2 receptors are components of epithelial cells and appear to be restricted to the ascending limb of the loop of Henle and to the collecting tubule. V2 receptors are coupled to adenyl cyclase and cyclic AMP production. While a great deal of information is available concerning vasopressin binding and action in various tissues, the receptor itself has not been characterized and the gene for the receptor as not been cloned. Difficulties in cloning the gene result from the lack of characterized receptor protein and no available antibodies to the receptor.
The specific aim of this proposal to clone the vasopressin receptor gene using alternative cloning strategies which do not necessarily require knowledge of the protein structure or availability of antibody to the receptor. These strategies use a mammalian transformed cell expression system which can be manipulated to produce and incorporate the vasopressin receptor into the plasma membrane. These cells are screened for expression of the receptor using various binding techniques. Following cloning of the vasopressin receptor, the DNA will be sequenced and amino acid sequence and structure will be determined. Using the initially isolated cDNA, the cDNA encoding the vasopressin receptor from other tissues will be isolated to provide comparison of variations in the vasopressin receptor. If cloning procedures yield success, large quantities of the protein can be produced and antibodies to the receptor developed. Finally, the cloned receptor gene will be used to study various disease states to determine if changes in receptor gene expression exist.
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