On co-transfecting human genomic DNA and a thymidine kinase hen into mouse tk- cells nad screening transformed cell lines for acquisition of receptors that would stimulate adenyl cyclase, we have isolated first a primary and then a secondary transformant cell line that expresses the human V2-type vasopressin receptor. The secondary transformant was designated HTB-2. Dot blots testing for human repetitive sequences in HTB-2 indicate that it contains less than 75 kb of human DNA, part of which encodes the vasopressin receptor. Established molecular biology techniques used in similar circumstances previously by others, should allow us to rescue the human V2 receptor gene, and with it clone its cDNA and deduce its primary structure. Thus, while efforts by many laboratories over the last 10 to 15 years have failed in attaining any significant purification of the V2 receptor protein, we are in a position to obtain long sought information about this receptor and use the cloned cDNA as a tool to investigate biochemical, physiological and physiopathological aspects of vasopressin action in body homeostasis.
The specific aims of the research proposed herein include: to rescue the human V2 vasopressin receptor gene from GTB-2 cells, to clone its cDNA and to deduce the amino acid sequence of this receptor; to investigate the molecular basis of familial nephrogenic diabetes insipidus, a disease that is characterized by lack of responsiveness to actions of vasopressin that are mediated by the V2 receptor cDNA that can be expected to be conserved as tools to clone other vasopressin receptors (Vla and Vlb) and the oxytocin receptor and determine the complexity of the human neurohypophyseal hormone receptors to determine by specific binding assays and effector stimulation assays (adenyl cyclase and phospholipase(s)) their pharmacologic and coupling properties; to map, initially in the rat and then as possible in human tissues, which cells express which neurohypophyseal hormone receptor.
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