Misfolded G Protein-Coupled Receptors and Endocrine Disease. Molecular Mechanisms and Therapeutic Prospects

Abstract

Misfolding of G protein-coupled receptors (GPCRs) caused by mutations frequently leads to disease due to intracellular trapping of the conformationally abnormal receptor. Several endocrine diseases due to inactivating mutations in GPCRs have been described, including X-linked nephrogenic diabetes insipidus, thyroid disorders, familial hypocalciuric hypercalcemia, obesity, familial glucocorticoid deficiency [melanocortin-2 receptor, MC2R (also known as adrenocorticotropin receptor, ACTHR), and reproductive disorders. In these mutant receptors, misfolding leads to endoplasmic reticulum retention, increased intracellular degradation, and deficient trafficking of the abnormal receptor to the cell surface plasma membrane, causing inability of the receptor to interact with agonists and trigger intracellular signaling. In this review, we discuss the mechanisms whereby mutations in GPCRs involved in endocrine function in humans lead to misfolding, decreased plasma membrane expression of the receptor protein, and loss-of-function diseases, and also describe several experimental approaches employed to rescue trafficking and function of the misfolded receptors. Special attention is given to misfolded GPCRs that regulate reproductive function, given the key role played by these particular membrane receptors in sexual development and fertility, and recent reports on promising therapeutic interventions targeting trafficking of these defective proteins to rescue completely or partially their normal function.

Keywords: G protein-coupled receptors; GPCR; GnRHR; gonadotropin receptors; gonadotropin-releasing hormone receptor; loss-of-function diseases; mutations in GPCRs; protein misfolding.

Figure 1

Schematic representation of the human GnRHR sequence, with circles representing amino acid residues. The red discontinuous lines within the light orange square, indicate the C14–C200 disulfide bridge, which helps to maintain the GnRHR in a conformation compatible with endoplasmic reticulum export and that is destabilized by the presence of lysine 191 (blue circle), as well as the C114–C196 bridge, which is a general structural requirement present in GPCRs belonging to the rhodopsin-like family. The light yellow square comprises the transmembrane domains (TMD) 4 and 5 which accommodates serine residues 168 and 217 that when replaced with arginine lead to severe, rescue-recalcitrant misfolding. The extracellular loop 1 (EL1) presents the conserved sequence Trp-Tyr-Ala-Gly (WYAG) [58]. In other rhodopsin-like GPCRs for peptides and biogenic amines, this sequence corresponds to the Trp-Xaa-Phe-Gly motif [(W/F)XφG, in which φ is a hydrophobic residue], which has been shown to be important for agonist-mediated receptor activation [62]. The light-blue square encompasses the sequence of the Catfish Ctail, which has been experimentally employed to test the role of the Ctail in the hGnRHR. Also indicated are the mutations E90K, Y108C, and T104I, whose effects on hGnRHR trafficking have been explored in detail [63]. The upper right inset describes some features of the mutations shown in the schematization of the receptor as well as of K191 and the Ctail, which is absent in type 1 GnRHR (see text for details).

Figure 2

Predicted structure of the upper-third portion of the hGnRHR based on homology modeling with the structure of bovine rhodopsin [76]. The antiparallel α-helices of transmembrane domains (TM) 1 to 7 are represented by the coiled structures. These TM domains are connected by the extracellular loops (EL) of the receptor (blue curved cords). Disulfide bonds between C14 and C200 (connecting the the NH₂-terminus and the EL2), as well as between C114 and C196 (at the COOH-terminal end of the EL1 and at the EL2) are shown as orange sticks. The location of the amino acid residues that represent a motif of four non-contiguous residues at positions L112 (at the EL1), Q208 (at the EL2), L300 (at the EL3), and D302 (at the EL3) that presumably control the destabilizing role of K191 (shown as purple, blue, and grey color sticks, at the EL2) on the association of the NH₂-terminus and the EL2 and subsequent formation of the C14–C200 disulfide bridge, are shown in colored circles and sticks (see the text and Refs. [64,69] for data and details).

Figure 3

Changes in sulfur-sulfur (S-S) distances between C14 and C200 in the hGnRHR when the C14–C200 is disrupted (||) in silico, in the presence and absence of K191. Superposition of the WT hGnRHR conformation (blue structures) and the final mutant conformations (grey structures) are shown. Lysine 191 and the C14–C200 and C114–C196 disulfide bridges of the WT hGnRHR are highlighted in blue (or orange in the mutants). Residues C14 and C200 are depicted in balls and sticks. S–S distances between residues in positions 14 and 200 are also indicated (dotted lines). Insets: distances (in nm) between S-S atoms as a function of time for 3 replicas. Observe that larger average distances are found in mutants bearing K191 (for original data and details see Ref. [76]).

Figure 4

Schematic representation of the human gonadotropín receptors showing the location of the inactivating mutations described to date (red and yellow circles). Mutations indicated by the yellow circles lead to misfolding and trafficking defective receptors with reduced or absent cell surface plasma membrane expression. Arbor-like structures (shaded in light green circles) represent potential glycosylation sites. (A) FSHR; (B) LHCGR.

Figure 5

In vitro and in silico features of the misfolded, trafficking-deficient, naturally occurring D408Y FSHR mutant and other laboratory-manufactured FSHRs with distinct substitutions at position 408 of the receptor protein. (a) Extra- and intracellular mean relative fluorescence intensity of the WT and D408Y FSHRs expressed in HEK-293 cells as disclosed by flow cytometry. Note the limited cell surface plasma membrane expression of the mutant receptor. The figure also shows data on the trafficking deficient A189V FSHR mutant included for comparison. (b) Representative Western blot of the WT FSHR and the D408Y, D408R, and D408A mutant FSHRs present in extracts of HEK-293 cells transiently transfected with each receptor species (lanes 3–6). The immunoblot shows the relevant portion of an autoradiogram in which the mature (mFSHR, ~80 KDa), plasma membrane-expressed, fully glycosylatyed form of the D408Y and D408A FSHRs are considerably reduced. These two latter FSHR mutants are predominantly detected as immature, intracellular forms of the receptor (iFSHR, KDa ≤ 75). Lane 1: WT FSHR from extracts of HEK-293 cells stably expressing the human FSHR; lane 2: Extracts from cells transfected with empty vector. (c) Dose-response curves for pSOMLuc expression by HEK-293 cells transiently cotransfected with the WT or mutant D408Y, D408R, or D408A FSHRs and the cAMP-sensitive pSOMLuc reporter plasmid, and exposed to increasing doses of recombinant human FSH (recFSH). Each point represents the mean ± SEM of 3 independent experiments. Note that the D408R and D408A FSHR mutants are completely or partially inactive when challenged with recFSH, whereas the function of the naturally occurring D408Y mutant also exhibits reduced activity albeit to a lesser extent than the other mutants. (d) Dynamic community analysis of the WT, D408Y, D480A, and D408R FSHRs. When compared with the WT FSHR, distinct connectivities among communities were observed in the mutant receptors. (e) Principal component analysis (PCA) to evaluate the collective motion of Cα atoms in the WT and D408Y, D408R, and D408A FSHRs. In these three-dimensional representations, it can be observed that the dynamics of the receptor disclosed differences in the amplitudes of interhelical domains (arrows) as a function of the mutation at position 408. See the text and Refs. [115,122] for data and details.