Rescue of Function of Mutant Luteinising Hormone Receptors with Deficiencies in Cell Surface Expression, Hormone Binding, and Hormone Signalling

Abstract

<b><i>Introduction:</i></b> G protein-coupled receptor (GPCR) mutations are implicated in many diseases. Most inactivating mutations cause receptor misfolding and prevent trafficking to the plasma membrane. Pharmacological chaperones can “rescue” cell surface expression of such mutants, presumably by stabilising correct folding of the nascent protein. <b><i>Objective:</i></b> Here we examine the scope of intracellularly retained luteinising hormone receptor (LHR) mutants that can be “rescued” by the pharmacological chaperone LHR-Chap, and whether this allosteric agonist can also restore the function of mutant LHRs with deficiencies in hormone binding or hormone-induced signalling. <b><i>Methods:</i></b> Mutant LHRs were expressed in HEK 293-T cells. Cell surface expression/localisation, hormone binding, and hCG/LHR-Chap signalling were determined by ELISA, radioligand binding, and inositol phosphate accumulation assays, respectively. Molecular modelling predicted LHR-Chap interactions. <b><i>Results:</i></b> LHR-Chap increased cell surface expression of a subset of retained mutants located in transmembrane helices predicted to be stabilised by LHR-Chap binding. For 3 (T461^3.47I, L502^4.61P, and S616^7.46Y) hCG-responsiveness was increased following treatment. LHRs with mutations in the hormone-binding site (C131^ECDR and I152^ECDT) or in the hinge region (E354^HingeK) had good cell surface expression but poor response to hormone stimulation, yet were responsive to allosteric activation by LHR-Chap.<b><i> Conclusions:</i></b> LHR-Chap, in addition to rescuing cell surface expression of intracellularly retained LHR mutants, can rescue function in mutant receptors with binding and signalling deficiencies that have normal cell surface expression. This demonstration of rescue of multiple elements of LHR dysfunction arising from inactivating mutations offers exceptional potential for treating patients with diseases arising from GPCR mutations in general.