Analysis of the V2 Vasopressin Receptor (V2R) Mutations Causing Partial Nephrogenic Diabetes Insipidus Highlights a Sustainable Signaling by a Non-peptide V2R Agonist

Abstract

Disease-causing mutations in G protein-coupled receptor (GPCR) genes, including the V2 vasopressin receptor (V2R) gene, often cause misfolded receptors, leading to a defect in plasma membrane trafficking. A novel V2R mutation, T273M, identified in a boy with partial nephrogenic diabetes insipidus (NDI), shows intracellular localization and partial defects similar to the two mutants we described previously (10). Although non-peptide V2R antagonists have been shown to rescue the membrane localization of V2R mutants, their level of functional rescue is weak. Interestingly, it has been reported that a non-peptide agonist, OPC51803, activates misfolded V2R mutants intracellularly without degradation, thus potentially serving as a therapeutic agent against NDI (14). In our current experiments, however, a peptide antagonist blocked arginine vasopressin (AVP)- or OPC51803-stimulated cAMP accumulation both in COS-7 and MDCK cells, suggesting that OPC51803 mainly stimulates cell surface V2R mutants. In addition, our analyses revealed that OPC51803 works not only as a non-peptide agonist that causes activation/β-arrestin-dependent desensitization of V2R mutants expressed at the plasma membrane but also as a pharmacochaperone that promotes the endoplasmic reticulum-retained mutant maturation and trafficking to the plasma membrane. The ratio of the pharmacochaperone effect to the desensitization effect likely correlates negatively with the residual function of the tested mutants, suggesting that OPC5 has a more favorable effect on the V2R mutants with a less residual function. We speculated that the canceling of the desensitization effect of OPC51803 by the pharmacochaperone effect after long-term treatment may produce sustainable signaling, and thus pharmacochaperone agonists such as OPC51803 may serve as promising therapeutics for NDI caused by misfolded V2R mutants.

Characterization of five novel vasopressin V2 receptor mutants causing nephrogenic diabetes insipidus reveals a role of tolvaptan for M272R-V2R mutation

“…A membrane permeable V2R antagonist named tolvaptan, previously known as OPC4, was shown to rescue several V2R NDI-causing mutants 29 – 31 . Treatment with tolvaptan improved the appearance of the complex-glycosylated band at 75 kDa in M272R mutant associated to a decrease of the immature 60–63 kDa band.…”

Cinacalcet corrects biased allosteric modulation of CaSR by AHH autoantibody

“…Immunoperoxidase staining and immunofluorescence imaging. Immunoperoxidase staining and immunofluorescence imaging were performed using patient or control sera (1:50-100 dilution) or a monoclonal antibody raised against amino acids 15-29 of CaSR (C0493, MilliporeSigma) ( Figure 1B) or 214-235 of CaSR (MA1-934, Affinity BioReagents) ( Figure 2B) or polyclonal anti-Flag antibody (F-7425, MilliporeSigma) ( Figure 2B) (31,72). Briefly, HEK293 cells expressing the human CaSR or Flag-tagged human CaSR were fixed in 100% methanol at -20°C for 2 minutes to evaluate whole-cell expression (with permeabilization).…”

“…To evaluate cell surface expression (without permeabilization), the cells were fixed at 4°C for 15 minutes in 4% paraformaldehyde/PBS. Bound IgG was detected by using either a peroxidase-conjugated, goat anti-human antiserum specific for the γ chain of IgG (074-1002, Kirkegaard and Perry Laboratories) from the AEC + system or goat anti-human IgG tagged with Alexa Fluor 488 (green) (A-11013, Molecular Probes), goat anti-mouse IgG tagged with Alexa Fluor 488 (green) (A-11029, Molecular Probes), or Alexa Fluor 594 (red) (A-11032, Molecular Probes), anti-rabbit IgG tagged with Alexa Fluor 488 (green) (A-11034, Molecular Probes), or Alexa Fluor 568 (red) (A-11036, Molecular Probes) as described previously (31,72). Fluorescence images were then collected by using an ECLIPSE E600 microscope (NIKON).…”

Succinic Semialdehyde Dehydrogenase Deficiency: An Update

“…In case of receptors (e.g. renal arginine-vasopressin V2 receptor the disease-causing variants of which are associated with Diabetes Insipidus), both agonists and antagonists have been shown to act as PCs [129,130]. Furthermore, folding-or oligomerization-defective nicotinic acetylcholine receptors and adrenergic receptors can be rescued by their respective ligands [131,132,133,134].…”