2004 Global Researcher Conference Proceeding

April 09 - 11, 2004

Conference: 2004 Global Researcher Conference
Title: Rescue of the Cell Surface Expression of Vasopressin V2 Receptor Mutants in Nephrogenic Diabetes Insipidus
Authors: Robben, Joris; Knoers, Nine; Deen, Peter M.T.
Institutions: University of Nijmegen, University Medical Centre Nijmegen

Nephrogenic Diabetes Insipidus (NDI) is a disorder in which patients are unable to concentrate their urine in response to the antidiuretic hormone arginine-vasopressin. In the majority of congenital NDI patients, the vasopressin V2 receptor (AVPR2) gene located on the X chromosome is mutated. Several V2R mutants identified in NDI were shown to be (partially) functional in vitro, but they are impaired in their export from the endoplasmic reticulum (ER), due to misfolding. Our goal is to study the molecular defects causing X-linked NDI as well as the effect of chemical chaperones on the routing of V2R mutants. For this, we stably-transfected Madin-Darby Canine Kidney (MDCK) cells, which are polarized cells derived from the collecting duct, with expression constructs encoding wild-type V2R, and the NDI causing AVPR2 mutants L44P, R113W, I130F, R137H, S167L, G201D, T204N and V206D. All receptors were fused to green fluorescent protein. Confocal analysis showed that the wt-V2R mainly co-localized with E-Cadherin in the basolateral membrane, and was internalized into late endosomes and lysosomes upon treatment with 1, 10 or 100 nM dDAVP for 45 minutes. The V2R mutants L44P, I130F, S167L and V206D co-localized with protein disulfide isomerase (PDI) in the ER, while the R113W, G201D and T204N mutants were also expressed to some extent in the basolateral plasma membrane. The R137H mutant was predominately present in late endosomes and lysosomes.

Treatment of the different cell lines with the chemical chaperones glycerol, DMSO, thapsigargin, tetra-methyl ammonium oxide, 4-phenyl-butyrate or growth at 27°C, did not rescue cell surface expression of the AVPR2 mutants, except for AVPR2-V206D, which was rescued by glycerol, DMSO and thapsigargin. Besides thapsigargin (which blocks ER Ca2+ pumps), rescue of AVPR2-V206D was also observed with ionomycin (allows extracellular Ca2+ entry), but neither one in the presence of the Ca2+-chelator BAPTA. Our data show that rescue of cell surface expression by chemical chaperones is dependent on the specific mutation in AVPR2 and that the rescuing effect of thapsigargin on AVPR2-V206D is likely due to an increased cytosolic instead of a reduced ER calcium level.

There are some V2R mutants that are able to perform their functions of binding with AVP and activating a signaling pathway that will lead to increased water reabsorption. However, as these mutants are misshapen, they cannot get free from the endoplasmic reticulum (ER) in order to get to the cell membrane, where they can bind to AVP. Robben, et al., tested eight different V2R mutants to see if any of a number of agents known as chemical chaperones could free the mutants from the ER and help them get to the membrane. Only one of the mutants, V2R-V206D, was capable of being rescued. The chaperones capable of performing this rescue were: glycerol, DMSO, thapsigargin and ionomycin. The researchers’ work found that rescue of mutated V2R by chemical chaperone is dependent of the specific mutation. Although the compounds tested can not be used in a clinical setting, increased insights in rescue of V2R mutants may eventually lead to identification of a cure for NDI.