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

Abstract

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 degrees 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.

Effects of Cellular, Chemical, and Pharmacological Chaperones on the Rescue of a Trafficking-defective Mutant of the ATP-binding Cassette Transporter Proteins ABCB1/ABCB4

“…Many of the chemical compounds that have been proposed are on the basis of the observation that function of the ER is intrinsically dependent on calcium concentrations. Thus, maintaining low calcium levels in the ER using the calcium pump inhibitor thapsigargin or other chemicals allows to correct abnormal protein trafficking of ⌬F508 CFTR (23,35) of certain mutants of the V2 vasopressin receptor (36) or of LQT2 channels (25). Another agent, 4-PB, is also able to functionally rescue a number of trafficdefective mutant membrane proteins, including ⌬F508 CFTR (22,33), mutants of the bone morphogenic protein receptor (27), of the epithelial sodium channel (37), the low-density lipoprotein receptor (26), ATP8B1 (38), and the ABC transporter ABCB11 responsible for biliary secretion of bile salts (24).…”

“…For instance, only six of 16 nephrin mutants were located at the cell surface after treatment with 4-PB (42). In the case of the vasopressin V2 receptor, only one of nine mutations showed improved maturation and plasma membrane rescue with glycerol and thapsigargin (36).…”

Biased Agonist Pharmacochaperones of the AVP V2 Receptor May Treat Congenital Nephrogenic Diabetes Insipidus

“…As in previous studies with antagonist pharmacochaperones, 11,19,20 we determined cNDI-hV 2 R activity after washing the cells (elimination of the pharmacochaperone) and subsequently stimulating them with AVP. First, optimal plasmid quantities were determined to avoid cNDI-hV 2 R plasma membrane expression and a cAMP response in control conditions (Supplemental Figure 1).…”

Anterograde Trafficking of G Protein-Coupled Receptors: Function of the C-Terminal F(X)6LL Motif in Export from the Endoplasmic Reticulum

“…HEK293T cells transfected with either ␣ 2B -AR or its mutants were allowed to express for 24 h before being cultured at 30°C or treated with 2% DMSO for an additional 24 h. Cell-surface expression of the receptors was then measured by intact cell ligand binding. Both treatment conditions have been shown to rescue the expression of certain mutant GPCRs (Bailey et al, 2004;Tan et al, 2004;Robben et al, 2006), presumably by increasing the conformational stability of the receptors, thereby facilitating their passage through the ER quality control system. Low-temperature culture (Fig.…”