During this past granting period we completed the molecular cloning of both the gene and the cDNA encoding the human antidiuretic hormone or type 2 vasopressin receptor (V2R). The complete procedure entailed the construction of a series of stable transformant cell lines expressing the V2 receptor encoded in human genomic DNA in murine L cells. A lambda EMBL3 phage containing the complete V2R transcription unit was identified by expression in L cells. A fragment of the EMBL3 insert (V2R gene), was used to clone the V2R cDNA from the transformed L cell and from a human kidney cDNA library. Both cDNAs encode the same protein of 371 amino acids that is predicted to have seven transmembrane domains characteristic of G protein coupled receptors. The coding region of the gene is interrupted by two short introns. We located the gene to the q28-qter portion of the X chromosome, coincidental with the locus of congenital nephrogenic diabetes insipidus (CNDI). Analysis of the genomic DNA from CNDI patients revealed the presence of mutations in the region encoding the receptor protein. The specific aims of the research proposed for the next five years are: l. To continue analyzing the mutations in V2R of newly identified families suffering CNDI. 2. To express the CNDI open reading frames (ORF) so as to determine which aspect of receptor function is altered: hormone binding and affinity, signal transduction, and receptor sequestration. As necessary studies on receptor targeting to the cell surface will be performed with epitope tagged receptors to evaluate the impact of cDNA modifications upon receptor protein expression. To investigate whether receptor sequestration can occur without phosphorylation. To complete the cloning of the human V1 receptor so as to construct V2R/V1R chimeras and delineate regions important for G protein coupling and receptor sequestration. 3. To identify the location of the extrarenal site(s) of expression of the V2R in the mouse by expression of the reporter gene beta-galactosidase introduced in the V2R gene by homologous recombination.