Mutations in G protein-coupled receptors that impact receptor trafficking and reproductive function

Abstract

G protein coupled receptors (GPCRs) are a large superfamily of integral cell surface plasma membrane proteins that play key roles in transducing extracellular signals, including sensory stimuli, hormones, neurotransmitters, or paracrine factors into the intracellular environment through the activation of one or more heterotrimeric G proteins. Structural alterations provoked by mutations or variations in the genes coding for GPCRs may lead to misfolding, altered plasma membrane expression of the receptor protein and frequently to disease. A number of GPCRs regulate reproductive function at different levels; these receptors include the gonadotropin-releasing hormone receptor (GnRHR) and the gonadotropin receptors (follicle-stimulating hormone receptor and luteinizing hormone receptor), which regulate the function of the pituitary-gonadal axis. Loss-of-function mutations in these receptors may lead to hypogonadotropic or hypergonadotropic hypogonadism, which encompass a broad spectrum of clinical phenotypes. In this review we describe mutations that provoke misfolding and failure of these receptors to traffick from the endoplasmic reticulum to the plasma membrane. We also discuss some aspects related to the therapeutic potential of some target-specific drugs that selectively bind to and rescue function of misfolded mutant GnRHR and gonadotropin receptors, and that represent potentially valuable strategies to treat diseases caused by inactivating mutations of these receptors.

Keywords: G protein-coupled receptors; Gonadotropin receptors; Gonadotropin-releasing hormone receptor; Hipogonadism; Intracellular trafficking; Pharmacological chaperones.

Figure 1

Intracellular trafficking of GPCRs from the ER to the plasma membrane and relation to the cellular quality control system. Newly synthesized proteins are translocated to the lumen of the ER where folding is both facilitated and/or corrected by molecular chaperones (oval structures). The correctly folded protein is thereafter exported to the Golgi complex for further processing (eg. glycosylation) (step 1 in clear circles) and routing to the plasma membrane. When folding fails, misfolded proteins are retained in the ER and eventually targeted for degradation through the polyubiquitination/proteasome pathway (step 2). Pharmacoperones (orange rhomboid structures) diffuse into the cell (step 3) and selectively bind the misfolded protein to correct misfolding promoting its export to the Golgi complex (step 4). Previously synthesized misfolded proteins, retained by the QCS, may still be rescued by pharmacoperones (steps 3 and 4 at the left). Mature processed proteins are then delivered to the cell surface PM (step 5), where the pharmacoperone can dissociate from the target protein (step 6) allowing the receptor to interact with agonist (step 7). Development of pharmacoperones that dissociate functions or rescue from agonism is currently needed as many pharmacoperone drugs are also receptor agonists/antagonists. Modified from Conn and Ulloa-Aguirre (2011), with permission of the publisher.

Figure 2

Dominant negative effect of the laboratory-manufactured R573A mutant hFSHR on Wt hFSHR PM expression (A and B) and function (C), and effect of co-transfecting the Wt hFSHR L526-V599 fragment (inset, black circles) cDNA on the dominant negative effects of the mutant. (A) Representative autoradiogram from an immunoblot of the hFSHR present in protein extracts from HEK-293 cells transfected with the cDNAs shown at the top of the blot. The autoradiograph was overexposed in order to show the expression levels of the mutant receptor species and the immature forms of the hFSHR. (B) HEK-293 cells were co-transfected with the cDNAs shown at the bottom of the graph and specific [¹²⁵I]-FSH binding was determined in the presence or absence of the L526-V599 fragment. Note that in contrast to the L526-V599 fragment, co-transfection with the hFSHR A607-N695 cDNA fragment (encoding the TM7 and the entire Ctail of the Wt receptor), did not affect expression of the Wt receptor co-transfected with the R573A mutant. (C) Maximal FSH-stimulated total cAMP accumulation in HEK-293 cells co-transfected with the cDNAs shown at the bottom of the graph; note that the L526-V599 fragment cDNA was co-transfected in increasing concentrations. The Wt and mutant hFSHR cDNAs were hosted by the pSG5 vector whereas the hFSHR fragments were in pcDNA3.1. The results shown in B and C are the mean ± SEM from three independent experiments. *p < 0.05 vs all other conditions; ^¶p < 0.01 vs all other conditions; ^§p < 0.05 vs Wt + L526-V599 fragment + empty vector. (m)FSHR, mature form of the hFSHR; (i)FSHR, immature form of the hFSHR. Co-transfection of the mutant and Wt hFSHRs resulted in decreased PM expression and function of the mature form of the hFSHR as well as reduced specific [¹²⁵I]-FSH binding. Co-transfection of the Wt and mutant FSHRs with the L526-V599 fragment led to almost complete functional recovery and PM expression of the hFSHR. Reproduced from Zariñán et al., (2010), with permission of Elsevier Ireland LTD.

Figure 3

Rescue of hLHR and hFSHR misfolded mutants by pharmacoperones. (A) Measurement of cAMP accumulation by CRE-luciferase reporter gene activaton after 24 h stimulation in cells expressing Wt (●), A593P mutant (■), or S616Y (▲) mutant hLHRs was determined over a range of concentrations of Org 42599. Data were fitted by sigmoidal dose-response curves with Hill coefficients of unity and are presented as fold versus basal values for each receptor (mean ± SEM from at least three independent experiments). (B) Org41841 rescues Wt hFSHR and mutant A189V (p= 0.002), but is unable to rescue other mutants with mutation at diverse sites in the molecule (SEMs shown). After transfection and incubation in Org41841, cellular response was assessed by a challenge with 100 ng hFSH. As mutants were obtained from different laboratories and were in different expression vectors, controls are shown with the corresponding Wt hFSHR and the empty vector in each case. Figure 3A is reproduced from Newton et al. (2011), with permission of the National Academy of Sciences USA; figure 3B is reproduced from Janovick et al. (2009) with permission of Elsevier Ireland Ltd.