1999 European Regional Conference Proceeding

May 12 - 16, 1999

Conference: 1999 European Regional Conference
Title: Nephrogenic diabetes insipidus in Italian families
Authors: Albertazzi, Elena; Barbier, Pascaline; Faranda, Sara; Frattini, Annalisa; Vezzoni, Paolo; Bettinelli, Alberto; Procaccio, Mirella; Chini, Bice
Institutions: 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, CNR Institute of Neuroscience
Bice Chini During the last three years, we carried on a study aimed at the identification of mutations of the vasopressin V2 receptor gene (AVPR2) in Italian patients affected by congenital Nephrogenic Diabetes Insipidus (NDI). Seventeen unrelated families with a clinical history of NDI have been investigated. In 14 of these families, we identified a mutation in the coding region of the V2R, a G-protein coupled receptor whose activation by AVP leads to water re-uptake. Nine of the identified mutations have not been described in the literature. Among these new mutations, four are predicted to produce a premature termination with a complete loss of function of the protein, and five are missense mutations, causing the exchange of a single amino acid residue. In one patient, we found the coexistence in the AVPR2 of two different nucleotide substitutions leading to two missense mutations. The first mutation is a C->T substitution at position 675 (expected aminoacidic change Arg204->Cys). The second is a G->A replacement at position 1126, which would cause an aminoacidic change in the C-terminal region of the receptor (Gly352->Asp). Two of the newly identified missense mutations were studied at the functional level: the W99R (Trp -> Arg) mutation involves a residue located in the first extracellular loop, a region known to be crucial in determining high affinity binding for agonists; the A84D (Ala -> Asp) mutation involves a residue that is adjacent to a very conserved aspartic acid located in the second transmembrane domain and known to participate in the process of receptor activation. Since our results indicate that at difference with the majority of AVPR2 mutants which are retained inside the intracellular compartments, the W99R and A84D receptors are able to reach the plasmamembrane where they can bind to the agonist (although with greatly reduced affinity) and become activated. These properties make them good candidates for pharmacological treatments aimed at increasing their sorting and/or permanence to the cell membrane. To this purpose, we treated cells transfected with the WT, W99R and A84D AVPR2 with chemical chaperones such as glycerol and DMSO at concentrations which were ineffective on cell viability. Our results indicate that chemical chaperones have no effect on the intracellular processing of these receptors.

Chini, et al., studied 17 unrelated Italian families with a clinical history of nephrogenic diabetes insipidus (NDI). The researchers identified mutations in the vasopressin-2 receptor (AVPR2) gene in 14 of the families. Nine of these mutations had not been previously described. Four of these new mutations are predicted to result in a complete loss of function of AVPR2 protein. The remaining five mutations are called missense mutations. This type of mutation causes a replacement of a single amino acid in the AVPR2 protein by an amino acid that is not normally at that point in the sequence of the amino acids that comprise the AVPR2 protein. Nonetheless, this single replacement greatly inhibits the function of the AVPR2s in which they occur. One patient had two missense mutations in his AVPR2 protein.

Chini, et al., used two of the newly identified mutations to see how the structural change in the AVPR2 protein affected its ability to function. The W99R mutation replaced a tryptophan amino acid with an arginine amino acid at amino acid position 99. Mutation A84D replaced an alanine amino acid with an aspartic acid amino acid at position 84. Unlike the majority of AVPR2 gene mutations which are retained inside intracellular compartments and not allowed to travel to their work site on the basolateral membrane, the W99R and A84D receptors are able to get to the basolateral membrane. (The membrane encircles the cell. The bottom and side sections of the membrane are called the basolateral membrane.) There they can bind to the antidiuretic hormone, vasopressin (although at a greatly reduced rate) and become activated.

The researchers tried treating these two mutants with chemical chaperones (proteins which help synthesize, fold and modify the AVPR2 within the cell) in an effort to restore their functional capacity, but the chaperones did not prove effective.