2000 Global Researcher Conference Proceeding

March 10 - 12, 2000

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Conference: 2000 Global Researcher Conference
Title: V2 vasopressin receptor function studied in mice and yeast
Authors: Yun, June; Erlenbach, Isolde; Kostenis, Evi; Schmidt, Clarice; Zhu, Xiangyang; Liu, Jie; Schulz, Angela; Schoneberg, Torsten; Pausch, Mark H.; Wess, Jurgen
Institutions: NIH, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Lab of Bioorganic Chemistry, National Institutes of Health, NIDDK, University of Maryland School of Medicine, University of Leipzig, NIH-NIDDK, Wyeth-Ayerst Research
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Wess

To generate a mouse model of human X-linked nephrogenic diabetes insipidus (XNDI), we used gene targeting technology to introduce the Glu242->Stop nonsense mutation into the mouse V2 vasopressin receptor (V2R) coding sequence. This mutation is known to cause XNDI in humans (Wildin et al., Am. J. Hum. Genet., 55, 266-277, 1994) and to lead to the synthesis of a truncated V2R protein that is functionally inactive (Schöneberg et al., EMBO. J., 15, 1283-1291, 1996). Crosses of female mice heterozygous for the V2R mutation with wild-type male littermates yielded V2R–deficient hemizygous male pups at the expected Mendelian ratio (25%). The male mutant pups displayed decreased urine osmolalities and an enlargement of renal pelvic space, failed to thrive, and died within the first week after birth due to hypernatremic dehydration. Daily s.c. injections of 5% dextrose solutions, either alone or in combination with hydrochlorothiazide and/or indomethacin, prolonged the lifetime of the V2R–deficient pups to about two weeks. In contrast to the male mutant mice, female mice heterozygous for the V2R mutation showed normal growth. However, these animals also displayed XNDI-like symptoms, characterized by reduced urine concentrating ability of the kidney, polyuria, and polydipsia. Western blot analysis showed that the complete or relative loss of functional V2Rs had no significant effect on the basal expression levels of aquaporin-2 and the bumetanide-sensitive Na-K-2Cl co-transporter (BSC-1). The V2R mutant mice described here should become useful tools for evaluating novel therapeutic approaches towards the treatment of XNDI.

To facilitate the structural and functional analysis of the V2R, we expressed the human V2R in Saccharomyces cerevisiae (baker’s yeast). The yeast strains used for this work were genetically modified, such that yeast growth was dependent on AVP-induced activation of the heterologously expressed V2R. Specifically, V2R stimulation resulted in the activation of the yeast pheromone pathway leading to the induction of a pheromone-sensitive HIS3 reporter gene and enabling V2R-expressing yeast cells to grow on medium lacking histidine. In the presence of AVP, the V2R coupled efficiently to a mutant version of the yeast G protein, Gpa1p, in which the C-terminal region was derived from mammalian G as (GPA1-Gas). In contrast, the efficiency of coupling to wild-type Gpa1p or a hybrid Gpa1p protein in which the C-terminal portion consisted of mammalian Gaq sequence (GPA1-Gaq) was greatly reduced. Interestingly, the Gq-coupled M3 muscarinic receptor, when expressed in yeast, interacted more efficiently with GPA1-Gaq than with GPA1-Gas. These observations indicate that the V2R is fully functional and retains its proper G protein selectivity in yeast. The system described here will allow the application of powerful genetic screens to gain novel insight into the structural determinants that govern V2R function. It can also be adapted to allow for screening for novel peptide ligands that can functionally rescue disease-causing mutant V2Rs.

To facilitate research into X-linked nephrogenic diabetes insipidus (XNDI), Yun, et al., generated a line of mice that bear a mutated vasopressin-2 receptor (V2R) gene. As a result, male mice carrying the mutant V2R gene die during the first week after birth. Female mice that carry a normal copy of the V2R gene as well as a copy of the mutant version survive into adulthood but show symptoms of XNDI.

The researchers also successfully expressed the human V2R in baker’s yeast. In this yeast strain, the V2R is fully functional. This system will allow researchers to investigate the structural elements of the V2R that underlie its function and to look for substances that can enable mutant V2Rs to carry out their functions similar to normal V2Rs.