Submitted on June 19, 2009
Revised on September 29, 2009
Accepted on October 5, 2009
Diverse Vasopressin V2 Receptor Functionality Underlying Partial Congenital Nephrogenic Diabetes Insipidus
Mia Faerch1*, Jane Hvarregaard Christensen2, Soren Rittig3, Jan-Ove Johansson4, Niels Gregersen1, Francis E de Zegher5, and Thomas Juhl Corydon2
1 Aarhus University Hospital, Skejby
2 Aarhus University
3 Aarhus University Hospital, Skejby Section
4 Sahlgrenska University Hospital
5 University of Leuven
* To whom correspondence should be addressed. E-mail: mia.faerch{at}ki.au.dk.
X-linked congenital nephrogenic diabetes insipidus (CNDI) is characterized by a defective renal response to the antidiuretic hormone (AVP) due to variations in the arginine vasopressin receptor 2 (AVPR2) gene. In a unique group of patients the renal insensitivity to the effects of AVP is incomplete resulting in a partial phenotype. To investigate the molecular defects, two previously published variations in the AVPR2 gene, known to cause a partial CNDI phenotype, were expressed in transiently transfected human embryonic kidney cells. One variation (p.Arg104Cys) is located in the first extracellular loop and the other variation (p.Ser329Arg) is located in the intracellular C-terminal of the receptor protein. Western blotting showed almost equal amounts of WT-V2R and Arg104Cys-V2R protein at steady state, whereas the level of Ser329Arg-V2R protein was lower. Confocal microscopy established that WT-V2R and Arg104Cys-V2R are localized on the cellular surface while the Ser329Arg-V2R primarily accumulates within the endoplasmic reticulum resulting in reduced surface expression. Ligand binding analysis demonstrated that the Bmax for cells expressing Arg104Cys-V2R and Ser329Arg-V2R were 14.8-fold and 2.5-fold lower than Bmax for WT-V2R, respectively. AVP affinity (1/Kd) for WT-V2R and the Ser329Arg-V2R was similar while 1/Kd for Arg104Cys-V2R was increased. cAMP assay revealed that cells expressing p.Arg104Cys-V2R or p.Ser329Arg-V2R produced 1.7-fold and 6.8-fold lower amounts of cAMP compared to WT-V2R, respectively. In conclusion, ligand binding and signal transduction capability is dependent on localization of the amino acid variation. Striking divergences, at the level of receptor functionality may thus underlie similar clinical phenotypes in CNDI.
