Dominant-Negative Action of Disease-Causing Gonadotropin-Releasing Hormone Receptor (GnRHR) Mutants: A Trait That Potentially Coevolved with Decreased Plasma Membrane Expression of GnRHR in Humans

Abstract

Loss of function by 11 of 13 naturally occurring mutations in the human GnRH receptor (hGnRHR) was thought to result from impaired ligand binding or effector coupling, but actually results from receptor misrouting. Homo- or heterodimerization of mutant receptors with wild-type (WT) receptors occurs for other G protein-coupled receptors and may result in dominant-negative or -positive effects on the WT receptor. We tested the hypothesis that WT hGnRHR function was affected by misfolded hGnRHR mutants. hGnRHR mutants were found to inhibit the function of WT GnRHR (measured by activation of effector and ligand binding). Inhibition varied depending on the particular hGnRHR mutant coexpressed and the ratio of hGnRHR mutant to WT hGnRHR cDNA cotransfected. The hGnRHR mutants did not interfere with the function of genetically modified hGnRHRs bearing either a deletion of primate-specific Lys(191) or the carboxyl-terminal tail of the catfish GnRHR; these show intrinsically enhanced expression. Moreover, a peptidomimetic antagonist of GnRH enhanced the expression of WT hGnRHR, but not of genetically modified hGnRHR species. The dominant-negative effect of the naturally occurring receptor mutants occurred only for the WT hGnRHR, which has intrinsic low maturation efficiency. The data suggest that this dominant negative effect accompanies the diminished plasma membrane expression as a recent evolutionary event.

Salt bridge integrates GPCR activation with protein trafficking

“…Most are misfolded and misrouted molecules (9) that are rescuable with pharmacoperones, such as In3 (12)(13)(14)(15)(16), and rerouted to the plasma membrane (10). In Fig.…”

“…Total IP was then determined (23). This assay has been validated as a sensitive measure of PME for functional receptors when expressed at low amounts of DNA (<100 ng per well) and stimulated by excess agonist (12,14,16,(24)(25)(26)(27)(28)(29)(30).…”

“…Twenty-three hours after transfection, the medium was replaced with 0.25 mL of fresh growth medium. Where indicated, pharmacoperones (indicated concentration) in 1% DMSO ("vehicle") were added for 4 h in respective media to the cells and then removed 18 h before agonist treatment (16). In this study, we used Trypan blue exclusion to show cell viability after drug exposure.…”

Refolding of misfolded mutant GPCR: Post-translational pharmacoperone action in vitro

“…Seventeen GnRHR point mutants have been identified from patients with human hypogonadotropic hypogonadism (HH; Leaños-Miranda et al, 2003;Leaños-Miranda et al, 2002;Ulloa-Aguirre et al, 2003;Ulloa-Aguirre et al, 2004) and all appear to exert their effect (i.e. loss or diminution of GnRH efficacy) by producing misfolded and misrouted receptors rather than by loss of the ability to bind ligand or couple to effector.…”

7TM Receptors

“…Alternatively, there is evidence for multiple active GnRHR conformations (Caunt et al, 2004;Maudsley et al, 2004;Millar et al, 2004) raising the possibility that type I GnRHR-mediated proliferation inhibition in hormone-dependent cancer cells is dependent upon different conformations (with different ligand specificity) than effects on G q/11 in pituitary cells . Loss-of-function mutations in the type I GnRHR and deficiency of GnRH I are associated with hypogonadotropic hypogonadism although some 'loss of function' mutations may actually prevent trafficking of 'functional' type I GnRHRs to the cell surface, as evidenced by recovery of function by nonpeptide antagonists (Leanos-Miranda et al, 2003). GnRHR signalling may be dependent upon receptor oligomerisation (Conn et al, 1982;Kroeger et al, 2001).…”