Rescue of misrouted GnRHR mutants reveals its constitutive activity

Abstract

G protein-coupled receptors (GPCR) play central roles in almost all physiological functions, and mutations in GPCR are responsible for over 30 hereditary diseases associated with loss or gain of receptor function. Gain of function mutants are frequently described as having constitutive activity (CA), that is, they activate effectors in the absence of agonist occupancy. Although many GPCR have mutants with CA, the GnRH receptor (GnRHR) was not, until 2010, associated with any CA mutants. The explanation for the failure to observe CA appears to be that the quality control system of the cell recognizes CA mutants of GnRHR as misfolded and retains them in the endoplasmic reticulum. In the present study, we identified several human (h)GnRHR mutants with substitutions in transmembrane helix 6 (F(272)K, F(272)Q, Y(284)F, C(279)A, and C(279)S) that demonstrate varying levels of CA after being rescued by pharmacoperones from different chemical classes and/or deletion of residue K(191), a modification that increases trafficking to the plasma membrane. The movement of the mutants from the endoplasmic reticulum (unrescued) to the plasma membrane (after rescue) is supported by confocal microscopy. Judging from the receptor-stimulated inositol phosphate production, mutants F(272)K and F(272)Q, after rescue, display the largest level of CA, an amount that is comparable with agonist-stimulated activation. Because mutations in other GPCR are, like the hGnRHR, scrutinized by the quality control system, this general approach may reveal CA in receptor mutants from other systems. A computer model of the hGnRHR and these mutants was used to evaluate the conformation associated with CA.

Fig. 1.

Saturation (A) and Scatchard (B) plots of binding of [¹²⁵I]-buserelin to hGnRHR mutants. Cos-7 cells were transfected with 25 ng of DNA for WT receptor or mutant as described in Materials and Methods. After rescue with In3 or by deletion of K¹⁹¹, Scatchard analysis was performed on all mutants as indicated except for C²⁷⁹A, C²⁷⁹S, Y²⁸⁴F, and Y²⁸⁴F-desK¹⁹¹, for which saturation binding was used, in which case the K_d was not determined (nd). The B_max and K_d values are from at least three independent experiments showing the variance between experiments as seen in Table 1. The inset to B shows mutant F²⁷²L that has not been rescued by pharmacoperone In3. The image marked B expanded shows the bottom portion of B, expanded for clarity.

Fig. 2.

Antagonist properties of pharmacoperones, In3, Q89, TAK-013, and A17777, assessed by inhibition of buserelin-stimulated IP production. Cos-7 cells were transfected with 10 ng of DNA for (A) hWT or (B) hWT-desK¹⁹¹ GnRHR as described in Materials and Methods. IP production was measured in response to 10⁻⁹ m Buserelin (a metabolically stable GnRH agonist) and in the presence of the indicated level of four different pharmacoperones as indicated. At least three independent experiments were performed in replicates of four to six. The optimal doses of pharmacoperones were selected from earlier studies (4).

Fig. 3.

CA of hGnRHR mutants rescued by pharmacoperones and assessed by agonist-independent IP production. IP production was assessed for mutants with K¹⁹¹ (the WT condition) (A) or without K¹⁹¹ (B). Cos-7 cells were transfected with 100 ng of WT (a control that lacks CA) or mutant cDNA as described in Materials and Methods. Mutants were incubated in media alone or rescued with each of four pharmacoperones; these drugs were then washed out, and IP production was measured in response to media alone (no agonist added). In all figures, sem are shown for least three independent experiments performed in replicates of four to six. The horizontal dashed line is IP production after transfection with vector only. Bars marked with an asterisk show values that differ from those obtained after transfection with vector only (two-way ANOVA). DMSO, Dimethylsulfoxide.

Fig. 4.

Agonist-stimulated IP production by mutants of the hGnRHR after pharmacoperone rescue. Functional activity was assessed for mutants with K¹⁹¹ (the WT condition) (A) or without K¹⁹¹ (B). Cos-7 cells were transfected with 10 ng of WT or mutant cDNA, with or without K¹⁹¹ as described in Materials and Methods. After rescue, IP production was measured in response to 10⁻⁷ m buserelin (metabolically stable GnRH agonist). In all figures, sem are shown for least three independent experiments performed in replicates of four to six.

Fig. 5.

Location of mutations in the hGnRHR. Helices are shown by cartoons colored blue (inactive conformation) and yellow (active conformation). Substituted residues are shown by sticks, highlighted by dots, and colored purple (inactive conformation) and orange (active conformation). A, Positions of residues mutated in this work (E⁹⁰K, F²⁷²Q, K, C²⁷⁹A, S, Y²⁸⁴F, and ΔK¹⁹¹) in inactive and active conformations of hGnRHR. Side chains of substituted residues (E⁹⁰, F²⁷², C²⁷⁹, and Y²⁸⁴) are shown by balls colored purple (for the inactive state) and orange (for the active state). K¹⁹¹ from the EL2 and proximal N¹⁸ from the N terminus are shown by sticks colored blue. B, Suggested changes in the ligand binding pocket on receptor activation. Positions of mutated residues (C²⁷⁹ and Y²⁸⁴) in the binding pocket of active (yellow) and inactive (blue) receptor conformations. Helix shift and rotamer change of Y²⁸⁴ and C²⁷⁹ during the receptor activation bring Y²⁸⁴ to the nonpolar lipid environment and C²⁷⁹ to the polar environment of the ligand binding pocket. Note the contraction of the ligand binding pocket in the active state caused by the inward movement of TM5, TM3, and TM7. C–J, Interactions between substituted residues in position 272 from the moving TM6 and adjacent residues from TM3, TM5, and TM7 in the model of the inactive and activated hGnRHR. Environment of F²⁷² of hGnRHR (C and G) and its substitutions, F²⁷²L (D and H), F²⁷²Q (E and I), and F²⁷²K (F and J), in inactive (C–F, blue) or active (G–J, yellow) receptor states.