Pharmacological chaperone activity of SR49059 to functionally recover misfolded mutations of the vasopressin V1a receptor.

Pharmacological chaperones represent a new class of ligand with the potential to facilitate the delivery of misfolded, but still active, G-protein-coupled receptors to the cell surface. Using transfected HEK 293T cells, treatment with a nonpeptide antagonist, SR49059, dramatically increased ( approximately 60-fold) the surface expression of a misfolded, nonfunctional and intracellularly localized vasopressin V(1a) receptor (V(1a)R) mutant (D148A). This rescue of surface expression (111 +/- 7%) was almost identical to wild type assessed by confocal microscopy and quantitative enzyme-linked immunosorbent assay-based techniques. Recovery was not specific to D148A, since other surface-impaired mutations, D148N and D148E, and wild type were also increased following SR49059 exposure. However, surface delivery was specific to SR49059, since V(1a)R-selective peptide ligands or unrelated ligands were unable to mimic this action, suggesting that SR49059 acts intracellularly. SR49059-mediated surface rescue was time-, mutant-, and concentration-dependent but not directly related to its binding affinity. Maximal recovery was achieved following 12 h of treatment and did not involve de novo receptor synthesis or a consequence of preventing endogenous constitutive activity and/or internalization. Once at the surface, all mutants displayed enhanced signaling ability, and D148A was able to undergo agonist-mediated internalization. SR49059 was not effectively removed from the receptor, since signaling (EC(50)) of both wild type and D148A was reduced approximately 40-fold. This is the first report of a pharmacological chaperone ligand to act on misfolded mutant V(1a) Rs. This work provides an excellent model to understand the mechanistic action of an important new class of drug that may have potential in the treatment of diseases caused by inherited mutations.