Functional rescue of misfolded vasopressin V1A receptor mutations using a pharmacological chaperone ligand

Stuart R. Hawtin. Institute of Cell Signalling, University of Nottingham, Queen’s Medical Centre, Nottingham, NG7 2UH, UK.

Pharmacological chaperones represent a new class of ligand with the potential to facilitate the delivery of misfolded, but still active, GPCRs to the cell surface. A mutation (D148A) within the highly conserved “DRY” triplet of the vasopressin V1a receptor (V1aR) was recently identified as misfolded, non-functional and not expressed on the cell surface (Hawtin, 2004). The aim of this study was to determine: (i) if ligands developed towards the V1aR act as pharmacological chaperones (ii), if they can rescue the surface expression of misfolded mutant receptors and if so (iii), the conditions required to do this and (iv), if their pharmacology is normal once at the cell surface.

In transfected HEK293T cells, pre-treatment with a non-peptide antagonist (SR49059; 10 µM for 20 hr) was found to rescue the surface expression of (D148A) to levels (111±7 %; n=5) almost identical to wild-type (Wt) using a quantitative ELISA-based assay. This recovery of cell surface expression was not specific to (D148A) as other mutations (D148N and D148E; each with impaired surface expression of 19±7 % and 43±4 % respectively) including Wt was also increased (94±8 %, n=4; 95±6 %, n=4; and 122±4 %, n=6; respectively) relative to Wt without drug. This increased surface expression was specific to SR49059 as non-cell permeable V1aR-selective peptide ligands were unable to mimic this action, thus suggesting that SR49059 acts intracellularly. SR49059 was able to increase surface expression that was time-, mutant-, and concentration-dependent. The concentration (EC50) to mediate 50 % recovery was different for each mutant with values of 1100±100 nM (n=4); 420 ±70 nM (n=3) and 190 ±80 nM (n=3) for D148A, D148N and D148E respectively. SR49059 also increased surface expression that reached maximal levels after 12 h for all mutants. The time (t1/2) required to achieve a 50 % recovery was 3.7±0.3 hr, n=4; 2.9 ±0.2 hr, n=3; and 2.4 ±0.1 hr, n=3; for D148A, D148N and D148E respectively. This chaperone-mediated increase in expression did not involve up-regulation of newly synthesized receptor, as pretreatment with cycloheximide did not prevent trafficking of mutants to the surface. Furthermore, this activity was not due to accumulation of receptor at the surface by preventing constitutive internalisation, as no increased expression was observed in the presence of the inhibitor concanavalin A. Once at the cell surface, these rescued mutants were able to signal and undergo agonist-mediated internalisation.

This is the first report of a pharmacological chaperone ligand to act on misfolded mutant V1aRs. This work provides novel and important insights into understanding the action of an important new class of drug which have the potential in the treatment of diseases caused by inherited mutations that result in abnormal receptor localisation such as cystic fibrosis and nephrogenic diabetes insipidus.

Hawtin, S.R (2004) Proceedings of the British Pharmacological Society at http://www.pa2online.org/Vol2Issue4abst 094P.html

This work is funded by a fellowship awarded by the University of Nottingham.