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Vol. 17, Issue 1, 379-386, January 2006

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Rescue of Vasopressin V2 Receptor Mutants by Chemical Chaperones: Specificity and Mechanism

J. H. Robben ^*, M. Sze ^*, N.V.A.M. Knoers , and P.M.T. Deen ^*

^* Department of Physiology, Nijmegen Centre for Molecular Life Sciences, Nijmegen, The Netherlands; Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

Submitted June 29, 2005; Revised October 20, 2005; Accepted October 21, 2005
Monitoring Editor: Jeffrey Brodsky

Because missense mutations in genetic diseases of membrane proteins often result in endoplasmic reticulum (ER) retention of functional proteins, drug-induced rescue of their cell surface expression and understanding the underlying mechanism are of clinical value. To study this, we tested chemical chaperones and sarco(endo)plasmic reticulum Ca²⁺ ATPase pump inhibitors on Madin-Darby canine kidney cells expressing nine ER-retained vasopressin type-2 receptor (V2R) mutants involved in nephrogenic diabetes insipidus. Of these nine, only V2R-V206D showed improved maturation and plasma membrane rescue with glycerol, dimethyl sulfoxide (DMSO), thapsigargin/curcumin, and ionomycin but not with other osmolytes or growth at 27°C. This revealed that rescue is mutant specific and that this mutant is prone to rescue by multiple compounds. Rescue did not involve changed expression of molecular chaperones calnexin, heat-shock protein (HSP) 70, or HSP90. V2R antagonist SR121463B treatment revealed that V2R-V206D and V2R-S167T were rescued and matured to a greater extent, suggesting that the rescuing activity of a pharmacological versus chemical chaperone is broader and stronger. Calcium measurements showed that rescue of V2R-V206D by thapsigargin, curcumin, and ionomycin was because of increased cytosolic calcium level, rather than decreased endoplasmic reticulum calcium level. The molecular mechanism underlying rescue by DMSO, glycerol, and SR121463B is different, because with these compounds intracellular calcium levels were unaffected.

Abbreviations used: 4-PBA, 4-phenyl butyric acid; [Ca²⁺]_cyt, cytosolic calcium level; [Ca²⁺]_ER, endoplasmic reticulum calcium level; CFTR, cystic fibrosis transmembrane conductance regulator; CLSM, confocal laser scanning microscopy; DMSO, dimethylsulfoxide; NDI, nephrogenic diabetes insipidus; HSP, heat-shock protein; ICC, immunocytochemistry; TMAO, tri-methyl-amine-N-oxide; V2R, vasopressin V2 receptor.

Address correspondence to: Peter M.T. Deen (p.deen{at}ncmls.ru.nl).

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