Abstract
Gonadotropin-releasing hormone (GnRH) receptor mutants from patients with hypogonadotropic hypogonadism are frequently misrouted proteins that exert a dominant-negative (DN) effect on human (h) wild-type (WT) receptor, due to oligomerization and retention in the endoplasmic reticulum. Pharmacologic chaperones restore correct folding, rescuing mutants and WT receptor from this oligomer. Rat WT retains the ability to oligomerize (since human and mouse mutants exert a DN effect on rat (r) WT sequence) but, unlike human or mouse, escapes the DN effect of GnRH receptor (Gn-RHR) mutants because rGnRHR mutants route to the plasma membrane with higher efficiency than mouse or human mutants. These distinct behaviors of mouse and rat GnRHRs (distinguished by only four semi- or non-conservative amino acid differences) led us to assess the role of each amino acid. The difference in both routing and the DN effect appears mediated primarily by Ser(216) in the rGnRHR. The homologous amino acid in the hGn-RHR is also Ser and is compensated for by the primate-unique insertion of Lys(191) that, alone, dramatically decreases routing of the receptor. These studies establish the relation between the DN effect and altered receptor trafficking and explain why hGnRHR is more susceptible to defective trafficking by disease-related point mutations than rodent counterparts.
Highlights
The gonadotropin-releasing hormone (GnRH)1 receptor (GnRHR) is a heptahelical G protein-coupled receptor found in the plasma membrane of pituitary gonadotropes [1, 2]
Structural Features of the GnRH receptor (GnRHR)—Fig. 1A is a graphic of the rat WT GnRHR sequence, indicating the four positions of semi- or non-conservative mutations between the rat and mouse sequence
The human WT GnRHR contains an “extra” amino acid, Lys191; this amino acid is absent in all pre-primate species sequenced to date
Summary
GnRH, gonadotropin-releasing hormone; GnRHR, gonadotropin-releasing hormone receptor; DN, dominant-negative; HH, hypogonadotropic hypogonadism; WT, wild-type; h, human; m, mouse; r, rat; DMEM, Dulbecco’s modified Eagle’s medium; Buserelin, D-tert-butyl-Ser6-des-Gly10-Pro9-ethylamide-GnRH; IN3, (2S)-2-[5-[2-(2-azabicyclo[2.2.2]oct-2-yl)-1,1-dimethyl-2-oxoethyl]-2(3,5-dimethylphenyl)-1H-indol-3-yl]-N-(2-pyridin-4-ylethyl)propan-1amine; IP, inositol phosphate. The cellular etiology of this effect is a stable physical interaction between the mutant and wild-type GnRHR, producing an oligomer that is recognized by the cellular quality control apparatus as a defective complex [25]. This oligomer is retained by the endoplasmic reticulum presumably degraded (or recycled) [11, 25]; plasma membrane expression of the WT receptor is thereby diminished. Like defective routing of mutants, this effect is superseded by pharmacoperones that rescue the mutant GnRHRs. Rodent GnRHRs (327 amino acids), which are normally transferred to the plasma membrane with higher efficiency than primate GnRHRs, lack this amino acid insertion. Role of this primate-specific modification was examined with regard to the amino acid that governs the highly efficient transfer of rat WT GnRHR to the plasma membrane and loss of the DN effect of its mutants
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