1998 Global Conference Proceeding

March 02 - 04, 1998

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Conference: 1998 Global Conference
Title: Mutations of the vasopressin V2 receptor gene in X-linked Nephrogenic Diabetes Insipidus: functional analysis of receptor mutants and identification of new mutations in Italian families.
Authors: Albertazzi, Elena; Zanchetta, Deborah; Faranda, Sara; Frattini, Annalisa; Vezzoni, Paolo; Appiani, A; Bettinelli, Alberto; Chini, Bice
Institutions: CNR Institute of Neuroscience, Consiglio Nazionale delle Ricerche Cellular and Molecular Pharmacology Center, Consiglio Nazionale delle Ricerche Institute of Advanced Biomedical Technologies, Istituto Tecnologie Biomediche Avanzate - ITBA, CNR-ITBA, University of Milan
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We have recently started a national study devoted to the identification of mutations of the V2 receptor gene in Italian patients affected by congenital Nephrogenic Diabetes Insipidus (NDI). Up to now, we have collected ten Italian families with a clinical history of congenital NDI. In eight NDI patients we were able to identify mutations in the coding region of the V2 receptor gene; of the eight mutations identified, six are novel mutations and two are recurrent mutations. In one patient, no mutations in the coding region of either the V2 or the aquaporin 2 gene (Bichet D. personal communication) were identified. The last family is currently under investigation.

Among the new mutations identified, four are predicted to produce frameshift and/or premature termination of the protein in the second intracellular loop, the fourth transmembrane region, the third extracellular region and the third intracellular loop respectively (deletion of 13 bp at bases 498-510; nonsense mutation at position 563; deletion of two nucleotides at position 207-208; deletion of one nucleotide at position 809). Since all these mutants are predicted to produce proteins lacking the correct number of transmembrane regions, a complete loss of function of these receptors is expected. Missense mutations were identified in four families. In two unrelated families, a recurrent mutation was identified at position 408. Finally, two new missense mutations were identified at position 322 (C->A) and at position 366 (T->C). In the first case, an Ala ->Asp substitution is produced at residue 84 (A84D), an aminoacid that is close to the highly conserved aspartate located in the second transmembrane domain. In the second case, the mutation induces a Trp ->Arg substitution in the first extracellular loop (W99R), a key region for agonist binding. The effects of these two new missense mutations on the function of the V2 receptor were then investigated.

After reproducing the two mutations by site-directed mutagenesis, we studied by immunofluorescence the cellular distribution of the mutant receptors in transiently transfected COS7 cells by using a monoclonal antibody directed against a myc epitope added to the N-terminal end of the receptor. Our results indicate that the A84D mutant is retained inside the intracellular compartments and is unable to reach the plasmamembrane in detectable amounts. On the contrary, the W99R mutant is able to reach the cell surface as well as the wild-type receptor. We thus carried out a pharmacological characterization of this receptor mutant by using both the tritited agonist vasopressin and an iodinated antagonist (a generous gift from C. Barberis). Our results indicate that mutation W99R impairs the binding of the two ligand to the receptor, confirming that residues located in the first extracellular loop play a key role in determining the ligand binding pocket of the receptor.

Supported by Telethon Grant E.391

Chini, et al., have studied the mutations in the V2 receptor genes of nine Italian families with a history of congenital nephrogenic diabetes insipidus (CNDI). In eight families, they identified mutations in a particular region (the coding region) of the V2 receptor gene. Six of these mutations had never been described in the literature before; two had been. In one family, no mutations were found in either the V2 or the aquaporin gene, the two most likely sites for NDI associated mutations.

Different types of mutations are likely to have different effects. The authors predicted that four of the new mutations would result in premature termination of the receptor protein's development in different areas of the V2 receptor gene, depending on the mutation. They predicted these mutations would result in a complete loss of function of the V2 receptor gene. The other four mutations, called missense mutations, changed the gene's genetic sequence so that it recognizes a different amino acid than it is supposed to. Two of these mutations had been previously recorded; two had not, the A84D mutant and the W99R mutant. The authors research indicated the A84D mutation rendered the V2 receptor incapable of reaching the cell wall, where it is supposed to go. The W99R mutant lets the V2 receptor reach the cell wall, but impairs its ability to bind hormones necessary for its proper functioning.