Human GnRH Receptor Research Articles

Mutations in the Human Gonadotropin-Releasing Hormone Receptor: Insights into Receptor Biology and Function

Pulsatile hypothalamic release of GnRH tightly controls reproduction by activating a specific cell membrane receptor (GnRHR) on the surface of pituitary gonadotrophs to stimulate luteinizing hormone and follicle-stimulating hormone secretion. During the last 10 years, 21 loss-of-function GNRHR mutations have been identified in patients with idiopathic hypogonadotropic hypogonadism. Although most patients with hypogonadotropic hypogonadism can be treated by delivery of exogenous pulsatile GnRH, in several cases, patients with GNRHR mutations fail to respond efficiently to GnRH treatment. The impact of each mutation on GnRHR function has been studied extensively in vitro, but correlation with clinical phenotype is not always evident. By combining recent advances into the molecular mechanisms controlling ligand binding and receptor activation with data accumulated from clinical studies, this review summarizes the overall structure-function relationships of the human GnRH receptor, with an emphasis on the impact of naturally occurring mutations. Furthermore, given that it was recently demonstrated that pharmacological chaperones can rescue altered receptor function in vitro, potential therapeutic approaches are also discussed.

Challenges and Opportunities of Trapping Ligands

Because gonadotropin-releasing hormone (GnRH) analogs constitute an important class of therapeutics for various reproductive and hormone-dependent disorders, many novel compounds have been discovered and studied. Several orally active nonpeptide GnRH antagonists have recently gained increased attention. In the study published in this issue of Molecular Pharmacology, Kohout et al. (p. 238) used small-molecule TAK-013 (sufugolix; developed previously by Takeda Chemical Industries) as a tool to elucidate the mechanism of its insurmountable antagonism. On the basis of receptor mutagenesis combined with molecular modeling, the authors hypothesized that certain amino acid sequences uniquely present in the human GnRH receptor amino terminus and extracellular loop 2 may form a "trap door" retarding dissociation of TAK-013. Such a trapping mechanism could be both ligand- and receptor species-specific. Although analogous models were previously proposed for other G protein-coupled receptors, the study by Kohout et al. (2007) provides an important advance in the GnRH antagonists field and an illustration of the fact that preclinical studies using animal models with nonhuman receptors may have very limited value in predicting drug efficacy in human disease. There are many examples showing that high-affinity protein, peptide, or nonpeptide agonists or antagonists have also enhanced clinical efficacy. However, there are also numerous studies indicating that very high receptor binding affinity is not a guarantee of drug efficacy and that other factors, including pharmacokinetic profile, ligand-induced receptor desensitization, and "trafficking," are critical in design and development of optimal drugs.

Selection, synthesis, and structure–activity relationship of tetrahydropyrido[4,3- d]pyrimidine-2,4-diones as human GnRH receptor antagonists

The present article describes a selection of a new class of small molecule antagonists for the h-GnRH receptor, their preparation, and evaluation in vitro. Three computational methods were combined into a consensus score, to rank order virtual templates. The top 5% of templates were further evaluated in silico and assessed for novelty and synthetic accessibility. The tetrahydropyrido[4,3- d]pyrimidine-2,4-dione core was selected for synthesis and evaluated in vitro. Using an array approach for analog design and synthesis, we were able to drive the binding below 10 nM for the h-GnRH receptor after two rounds of optimization.

Synthesis and evaluation of homo-bivalent GnRHR ligands

G protein coupled receptors (GPCRs) are important drug targets in pharmaceutical research. Traditionally, most research efforts have been devoted towards the design of small molecule agonists and antagonists. An interesting, yet poorly investigated class of GPCR modulators comprise the bivalent ligands, in which two receptor pharmacophores are incorporated. Here, we set out to develop a general strategy for the synthesis of bivalent compounds that are projected to bind to the human gonadotropin-releasing hormone receptor (GnRHR). Our results on the dimerisation of a known GnRHR antagonist, with as key step the Huisgen 1,3-cycloaddition, and their ability to bind to and antagonize GnRH-induced GnRHR stimulation, are presented here.

Structure−Activity Relationship Studies of Gonadotropin-Releasing Hormone Antagonists ContainingS-Aryl/Alkyl Norcysteines and Their Oxidized Derivatives

A series of acyline analogues incorporating l- and d-isomers of S-arylated/alkylated norcysteines [Ncy(R), where R is 2-naphthyl, methyl, and isopropyl] at positions 1, 4, 7, and 10 were synthesized. Some of these analogues were mono- and dioxidized to sulfoxides and sulfones. All of the analogues of acyline were screened for the antagonism of the GnRH-induced response in a reporter gene assay in HEK-293 cells expressing the human GnRH receptor. Nine of the analogues (9, 11, 15, 16, 17, 19, 20, 21, and 22) had antagonistic potency (IC50 < 2 nM) similar to that of acyline (IC50 = 0.52 nM) in this assay. Selected analogues (9, 11, 15, 16, 19, and 21) were tested in vitro for their antagonism at the rat GnRH-R in a reporter gene assay as well as in an in vivo intact male rat assay. Analogues 9 and 15 were the most potent in suppressing testosterone levels.

Suppression of Serum Luteinizing Hormone in Postmenopausal Women by an Orally Administered Nonpeptide Antagonist of the Gonadotropin-Releasing Hormone Receptor (NBI-42902)

Context: Parenteral administration of peptide GnRH analogs is widely used in clinical practice for the suppression of pituitary gonadotropins. NBI-42902 is an orally available, high-affinity nonpeptide antagonist of the human GnRH receptor. Objective: The objective was to evaluate the safety, pharmacokinetics, and inhibitory effects on gonadotropin secretion of NBI-42902 in postmenopausal women. Design: This was a phase I, double-blind, placebo-controlled, single-dose study with sequential dose escalation. Participants: Fifty-six healthy, postmenopausal women were included. FSH levels were greater than 40 IU/liter, and body mass index was within 20% of ideal values for all subjects. Interventions: Subjects were administered 5, 10, 25, 50, 75, 100, 150, or 200 mg NBI-42902 as an oral solution. Main Outcome Measures: Safety, tolerability, and serum LH and FSH concentrations were evaluated. Results: NBI-42902 was well tolerated. Serum LH concentrations rapidly declined, and dose-dependent suppression was obs...

Calnexin regulated gonadotropin-releasing hormone receptor plasma membrane expression

A significant proportion of human gonadotropin-releasing hormone receptors (GnRHRs) are normally retained in the endoplasmic reticulum (ER); however, nearly all rat GnRHRs are routed to the plasma membrane. When mutations are introduced into either receptor, considerably more of the proteins are recognized by the quality control system (QCS) as misfolded and retained compared with wild-type (WT) receptor, resulting in decreased signaling in the presence of agonist. Calnexin, a component of the QCS, decreased plasma membrane expression of the GnRHRs, an effect that was mediated by a physical interaction between the receptor and the calnexin. Only the human receptor showed reduced signaling because it had fewer spare receptors compared with the rat GnRHR, allowing calnexin to affect signaling. Calnexin did not affect receptor signaling when K(191) was deleted from the human WT GnRHR. Removal of this amino acid decreases receptor misfolding and increases plasma membrane expression. K(191) is not present in the rat WT GnRHR. A pharmacological chaperone that corrects GnRHR misfolding, increased expression of the human WT GnRHR in the presence of calnexin. Calnexin apparently retains misfolded GnRHRs but routes correctly folded receptors to the plasma membrane. Mutation of a calnexin protein kinase C consensus phosphorylation site promoted increased retention of the human GnRHR, suggesting that calnexin phosphorylation controls the retention mechanism. We conclude that a proportion of the human and the rat WT GnRHR appears to be retained in the ER by calnexin, an effect that decreases GnRHR signaling capacity.

Intracellular gonadotropin-releasing hormone receptors in breast cancer and gonadotrope lineage cells

Gonadotropin-releasing hormone receptors (GnRHRs) are expressed in gonadotropes and several extra-pituitary sites. They are assumed to be cell surface proteins but the human (h) GnRHR lacks features favoring plasma membrane localization and receptor location varies with cell type. When expressed in mammary (MCF7) cells, cell surface hGnRHR binding was much lower than that of mouse and sheep GnRHRs (type I GnRHRs without C-terminal tails), Xenopus (X) and marmoset type II GnRHRs (type II GnRHRs with C-tails) or chimeric receptors (type I GnRHRs with added XGnRHR C-tails). hGnRHR binding was higher in alphaT4 (gonadotrope-derived) cells and was increased less by C-tail addition. Whole cell levels of tagged human, Xenopus and chimeric GnRHRs were comparable (Western blotting) and confocal microscopy revealed that the hGnRHR is primarily intracellular (distribution similar to the endoplasmic reticulum marker, calreticulin), whereas most XGnRHR is at the plasma membrane, and adding the C-tail increased cell surface hGnRHR levels. A membrane-permeant antagonist increased cell surface hGnRHR number (>4-fold, t1/2 = 4 h) and also increased hGnRHR signaling and hGnRHR-mediated inhibition of proliferation. A more rapid increase in hGnRHR binding occurred when the temperature was raised from 4 to 37 degrees C (>5-fold, t1/2 = 15 min) and this effect was prevented by mutation to prevent signaling. Thus, cell surface GnRHR expression depends on receptor and cell type and the hGnRHR is primarily an intracellular protein that traffics to the cell surface for signaling in MCF7 cells. Manipulations favoring such trafficking may facilitate selective targeting of extra-pituitary GnRHRs.

Allosteric and Orthosteric Binding Modes of Two Nonpeptide Human Gonadotropin-Releasing Hormone Receptor Antagonists

Nonpeptide antagonists of the human gonadotropin-releasing hormone receptor (GnRH-R) have been the subject of considerable interest because of their potential as a new class of oral therapeutics for the treatment of sex hormone-dependent diseases and infertility. While many classes of competitive GnRH-R antagonists have been described, we present here the first characterization of an allosteric nonpeptide GnRH-R antagonist. Previously, 5-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-ylmethyl)furan-2-carboxylic acid (2,4,6-trimethoxyphenyl)amide (here called Furan-1) had been demonstrated to be a potent GnRH-R antagonist both in vitro and in vivo. Using mutagenesis, the binding sites for Furan-1 and another potent nonpeptide antagonist (NBI-42902) have been mapped and are shown to be adjacent but nonoverlapping. Furan-1 is shown to affect the binding kinetics of radiolabeled peptide agonists as well as radiolabeled NBI-42902, and the kinetic data fit the allosteric ternary complex model. Furan-1 is considerably negatively cooperative with the nonpeptide antagonist and extremely negatively cooperative with the peptide agonist [125I-His5,d-Tyr6]GnRH so that it is nearly indistinguishable from an orthosteric competitive compound. Taken together, these data were used to develop a model of the nonpeptides bound to the GnRH-R binding site consistent with the current data.

Identification of a gonadotropin-releasing hormone receptor orthologue in Caenorhabditis elegans

BackgroundThe Caenorhabditis elegans genome is known to code for at least 1149 G protein-coupled receptors (GPCRs), but the GPCR(s) critical to the regulation of reproduction in this nematode are not yet known. This study examined whether GPCRs orthologous to human gonadotropin-releasing hormone receptor (GnRHR) exist in C. elegans.ResultsOur sequence analyses indicated the presence of two proteins in C. elegans, one of 401 amino acids [GenBank: NP_491453; WormBase: F54D7.3] and another of 379 amino acids [GenBank: NP_506566; WormBase: C15H11.2] with 46.9% and 44.7% nucleotide similarity to human GnRHR1 and GnRHR2, respectively. Like human GnRHR1, structural analysis of the C. elegans GnRHR1 orthologue (Ce-GnRHR) predicted a rhodopsin family member with 7 transmembrane domains, G protein coupling sites and phosphorylation sites for protein kinase C. Of the functionally important amino acids in human GnRHR1, 56% were conserved in the C. elegans orthologue. Ce-GnRHR was actively transcribed in adult worms and immunoanalyses using antibodies generated against both human and C. elegans GnRHR indicated the presence of a 46-kDa protein, the calculated molecular mass of the immature Ce-GnRHR. Ce-GnRHR staining was specifically localized to the germline, intestine and pharynx. In the germline and intestine, Ce-GnRHR was localized specifically to nuclei as revealed by colocalization with a DNA nuclear stain. However in the pharynx, Ce-GnRHR was localized to the myofilament lattice of the pharyngeal musculature, suggesting a functional role for Ce-GnRHR signaling in the coupling of food intake with reproduction. Phylogenetic analyses support an early evolutionary origin of GnRH-like receptors, as evidenced by the hypothesized grouping of Ce-GnRHR, vertebrate GnRHRs, a molluscan GnRHR, and the adipokinetic hormone receptors (AKHRs) and corazonin receptors of arthropods.ConclusionThis is the first report of a GnRHR orthologue in C. elegans, which shares significant similarity with insect AKHRs. In vertebrates, GnRHRs are central components of the reproductive endocrine system, and the identification of a GnRHR orthologue in C. elegans suggests the potential use of C. elegans as a model system to study reproductive endocrinology.

Pharmacological Characterization of a Novel Nonpeptide Antagonist of the Human Gonadotropin-Releasing Hormone Receptor, NBI-42902

Suppression of the hypothalamic-pituitary-gonadal axis by peptides that act at the GnRH receptor has found widespread use in clinical practice for the management of sex-steroid-dependent diseases (such as prostate cancer and endometriosis) and reproductive disorders. Efforts to develop orally available GnRH receptor antagonists have led to the discovery of a novel, potent nonpeptide antagonist, NBI-42902, that suppresses serum LH concentrations in postmenopausal women after oral administration. Here we report the in vitro and in vivo pharmacological characterization of this compound. NBI-42902 is a potent inhibitor of peptide radioligand binding to the human GnRH receptor (K(i) = 0.56 nm). Tritiated NBI-42902 binds with high affinity (K(d) = 0.19 nm) to a single class of binding sites and can be displaced by a range of peptide and nonpeptide GnRH receptor ligands. In vitro experiments demonstrate that NBI-42902 is a potent functional, competitive antagonist of GnRH stimulated IP accumulation, Ca(2+) flux, and ERK1/2 activation. It did not stimulate histamine release from rat peritoneal mast cells. Finally, it is effective in lowering serum LH in castrated male macaques after oral administration. Overall, these data provide a benchmark of pharmacological characteristics required for a nonpeptide GnRH antagonist to effectively suppress gonadotropins in humans and suggest that NBI-42902 may have clinical utility as an oral agent for suppression of the hypothalamic-pituitary-gonadal axis.

G-Protein-Coupled Receptor Trafficking: Understanding the Chemical Basis of Health and Disease

The primary function of cell surface receptors is to recognize specific chemical signals from other substances and produce a biological response. Point mutations in cell surface receptors may result in production of misfolded proteins that are translated but do not reach their proper functional destination in the cell. Also, for some G-protein-coupled receptors, large amounts of wild-type receptor may be destroyed without arriving at the plasma membrane (PM). For the human gonadotropin-releasing hormone receptor, this "inefficiency" has resulted from strong and convergent evolutionary pressure, producing receptor molecules that are sensitive to single changes in chemical charge and are delicately balanced between expression at the PM or retention/degradation in the endoplasmic reticulum. This Perspective focuses on the evolved mechanisms that control PM expression of this receptor at this post-translational level.

Determination of the Binding Mode of Thienopyrimidinedione Antagonists to the Human Gonadotropin Releasing Hormone Receptor Using Structure−Activity Relationships, Site-Directed Mutagenesis, and Homology Modeling

We have investigated the specific interactions of a series thienopyrimidinediones with the gonadotropin-releasing hormone receptor (GnRH-R). Competitive radioligand binding assays were used to determine the effect of several mutants on nonpeptide binding. Distinct interactions were observed in two separate regions: the N-terminal end of TM7 and the C-terminal end of TM6. The effects of mutants at D302((7.32)) and H306((7.36)) suggest that these residues are part of a hydrogen-bond network important for anchoring the nonpeptides. Structure-activity relationships indicated urea substituents on the 6-(4-aminophenyl) group with a trans conformational preference bind with high affinity and are sensitive to D302((7.32)) mutations. Another interaction area was found between the N-benzyl-N-methylamino substituent and L300((6.68)) and Y290((6.58)). These interaction sites facilitated the derivation of a model in which a representative member of the series was docked into GnRH-R. The model is consistent with known SAR and illuminates inconsistencies with previous hypotheses regarding how this series interacts with the receptor.

Model of gonadotropin-releasing hormone and gonadotropin-releasing hormone complex

Gonadotropin-releasing hormone (GnRH) neurons provide the primary driving force upon the other levels of the reproductive axis. Similar to other hormone, GnRH receptor can be identified. The main intracellular pathway activated by stimulation of GnRH receptors (GnRHRs) is related to Gq protein which induces a cytoplasmic metabolic cascade. The structures of both GnRH and GnRHR have been documented inn the recent year. However, the knowledge on the complex, recombination, between GnRH and GnRHR is limited. Here, the author performs an analysis to study the molecular structure of human GnRH and GnRHR complex. To reach the target, a computer-based study for protein recombination modeling is performed.

Kinetics of nonpeptide antagonist binding to the human gonadotropin-releasing hormone receptor: Implications for structure–activity relationships and insurmountable antagonism

Numerous nonpeptide ligands have been developed for the human gonadotropin-releasing hormone (GnRH) receptor as potential agents for treatment of disorders of the reproductive-endocrine axis. While the equilibrium binding of these ligands has been studied in detail, little is known of the kinetics of their receptor interaction. In this study we evaluated the kinetic structure–activity relationships (SAR) of uracil-series antagonists by measuring their association and dissociation rate constants. These constants were measured directly using a novel radioligand, [ 3H] NBI 42902, and indirectly for unlabeled ligands. Receptor association and dissociation of [ 3H] NBI 42902 was monophasic, with an association rate constant of 93 ± 10 μM −1 min −1 and a dissociation rate constant of 0.16 ± 0.02 h −1 ( t 1/2 of 4.3 h). Four unlabeled compounds were tested with varying substituents at the 2-position of the benzyl group at position 1 of the uracil (–F, –SO(CH 3), –SO 2(CH 3) and –CF 3). The nature of the substituent did not appreciably affect the association rate constant but varied the dissociation rate constant >50-fold ( t 1/2 ranging from 52 min for –SO(CH 3) to >43 h for –CF 3). This SAR was poorly resolved in standard competition assays due to lack of equilibration. The functional consequences of the varying dissociation rate were investigated by measuring antagonism of GnRH-stimulated [ 3H] inositol phosphates accumulation. Slowly dissociating ligands displayed insurmountable antagonism (decrease of the GnRH E max) while antagonism by more rapidly dissociating ligands was surmountable (without effect on the GnRH E max). Therefore, evaluating the receptor binding kinetics of nonpeptide antagonists revealed SAR, not evident in standard competition assays, that defined at least in part the mode of functional antagonism by the ligands. These findings are of importance for the future definition of nonpeptide ligand SAR and for the identification of potentially useful slowly dissociating antagonists for the GnRH receptor.

Suppression of Serum Luteinizing Hormone in Postmenopausal Women by an Orally Administered Nonpeptide Antagonist of the Gonadotropin-Releasing Hormone Receptor (NBI-42902)

Parenteral administration of peptide GnRH analogs is widely used in clinical practice for the suppression of pituitary gonadotropins. NBI-42902 is an orally available, high-affinity nonpeptide antagonist of the human GnRH receptor. The objective was to evaluate the safety, pharmacokinetics, and inhibitory effects on gonadotropin secretion of NBI-42902 in postmenopausal women. This was a phase I, double-blind, placebo-controlled, single-dose study with sequential dose escalation. Fifty-six healthy, postmenopausal women were included. FSH levels were greater than 40 IU/liter, and body mass index was within 20% of ideal values for all subjects. Subjects were administered 5, 10, 25, 50, 75, 100, 150, or 200 mg NBI-42902 as an oral solution. Safety, tolerability, and serum LH and FSH concentrations were evaluated. NBI-42902 was well tolerated. Serum LH concentrations rapidly declined, and dose-dependent suppression was observed. Maximal change from baseline LH concentrations ranged from -19 +/- 5% in the 5-mg group to -55 +/- 2% in the 150-mg group. Suppression of FSH was less pronounced (-15 to -22% of baseline). NBI-42902 was rapidly absorbed after oral administration with a terminal elimination half-life ranging from 2.7 +/- 0.3 to 4.8 +/- 0.8 h. A clear relationship between plasma NBI-42902 concentrations and LH suppression was evident. Dose-dependent LH suppression was achieved by oral administration of a nonpeptide GnRH antagonist suggesting that compounds such as NBI-42902 may enable adjustable gonadotropin suppression as part of novel treatment strategies for benign gynecological conditions.

Identification of new gonadotrophin-releasing hormone partial agonists

GnRH agonists or antagonists are currently utilized as therapeutic agents in a number of diseases. A side-effect of prolonged treatment with GnRH analogues is hypoestrogenism. In this study, we tested the in vitro potency of different GnRH analogues originally found to be partial agonists (i.e. analogues with decreased efficacy for activating or stimulating their cognate receptor) as well as novel analogues, to identify compounds that might potentially be useful for partial blockade of gonadotrophin release. Cultured COS-7 cells transiently expressing the rat or human GnRH receptor (GnRHR) were exposed to increasing concentrations (10(-8) to 10(-5) M) of GnRH analogues (c(4-10)[Asp4,DNal6,Dpr10]-GnRH; c(4-10) [Dpr4,DNal6,Asp10]-GnRH; c(4-10)[Cys(4,10),DNal6]-GnRH; c[Eaca1,DNal6]-GnRH; c[Gly1,DNal6]-GnRH; c[betaAla1,DTrp6]-GnRH; c[Dava1,DNal6]-GnRH; c[Gaba1, DNal6]-GnRH), and the ability of these analogues to provoke or antagonize GnRH-stimulated inositol phosphate production was assessed. With both human and rat GnRHRs, c[Eaca1,DNal6]-GnRH, c[Gly1,DNal6]-GnRH, c[betaAla1,DTrp6]-GnRH and c[Dava1,DNal6]-GnRH exhibited partial agonist activity (35-87% of the maximal efficacy shown by 10(-6) M GnRH), whereas c[Gaba1,DNal6]-GnRH behaved as a partial agonist with the human GnRHR and as full agonist with the rat GnRHR. c(4-10)[Asp4, DNal6,Dpr10]-GnRH and c(4-10)[Dpr4,DNal6,Asp10]-GnRH exhibited full antagonist activity with both GnRHRs, and c(4-10) [Cys(4,10),DNal6]-GnRH was a weak, partial agonist with the human GnRHR and a full antagonist with the rat GnRHR. With the exception of c[Gaba1,DNal6]-GnRH stimulation of the human GnRHR, and c[Dava1,DNal6]-GnRH and c[Gaba1, DNal6]-GnRH stimulation of the rat GnRHR, all partial agonists also exhibited antagonist activity in the presence of the exogenous full agonist. The results demonstrate that structurally similar analogues display variable potencies and efficacies in vitro for a specific GnRHR as well as for the human versus the rat GnRHR. Their ultimate in vivo usefulness to treat clinical conditions in which complete suppression of gonadotroph activity is not required remains to be investigated.

The LIM-Homeodomain Proteins Isl-1 and Lhx3 Act with Steroidogenic Factor 1 to Enhance Gonadotrope-Specific Activity of the Gonadotropin-Releasing Hormone Receptor Gene Promoter

The GnRH receptor (GnRH-R) plays a central role in mammalian reproductive function throughout adulthood. It also appears as an early marker gene of the presumptive gonadotrope lineage in developing pituitary. Here, using transient transfections combined with DNA/protein interaction assays, we have delineated cis-acting elements within the rat GnRH-R gene promoter that represent targets for the LIM-homeodomain (LIM-HD) proteins, Isl-1 and Lhx3. These factors, critical in early pituitary development, are thus also crucial for gonadotrope-specific expression of the GnRH-R gene. In heterologous cells, the expression of Isl-1 and Lhx3, together with steroidogenic factor 1 (SF-1), culminates in the activation of both the rat as well as human GnRH-R promoter, suggesting that this combination is evolutionarily conserved among mammals. The specificity of these LIM-HD factors is attested by the inefficiency of related proteins, including Lhx5 and Lhx9, to activate the GnRH-R gene promoter, as well as by the repressive capacity of a dominant-negative derivative of Lhx3. Accordingly, targeted deletion of the LIM response element decreases promoter activity. In addition, experiments with Gal4-SF-1 fusion proteins suggest that LIM-HD protein activity in gonadotrope cells is dependent upon SF-1 binding. Finally, using a transgenic model that allows monitoring of in vivo promoter activity, we show that the overlapping expression of Isl-1 and Lhx3 in the developing pituitary correlates with promoter activity. Collectively, these data suggest the occurrence of a specific LIM-HD pituitary code and designate the GnRH-R gene as the first identified transcriptional target of Isl-1 in the anterior pituitary.

Protein Folding as Posttranslational Regulation: Evolution of a Mechanism for Controlled Plasma Membrane Expression of a G Protein-Coupled Receptor

Recent studies reveal that a number of G protein-coupled receptors (GPCRs) and other proteins are expressed inefficiently at the site normally associated with their biological action. In the case of some GPCRs, large amounts of receptor (perhaps more than half) may be destroyed without ever binding ligand or even arriving at the plasma membrane. For the human GnRH receptor (GnRHR), this apparent inefficiency has evolved under strong and convergent evolutionary pressure. The result is a human GnRHR molecule that is delicately balanced between either expression at the plasma membrane (PM) or retention/degradation in the endoplasmic reticulum, an effect mediated by engagement with the cellular quality control system. This balance appears to be the reason that the human receptor, but not the rat or mouse counterpart (which are more robustly routed to the PM), is highly susceptible to single-point mutations that result in disease. A single change in net charge is sufficient to tip the balance in favor of the endoplasmic reticulum and diminish GnRHR available at the PM. The apparent paradox that results from observing convergent pressure for evolution of a receptor that is both inefficiently produced and highly susceptible to mutational disease suggests that this approach must offer a strong advantage. This review focuses on the evolved mechanisms and considers that this is an underappreciated mechanism by which the cell controls functional levels of receptors and other proteins at the posttranslational level.

Regulation of G Protein-coupled Receptor Trafficking by Inefficient Plasma Membrane Expression: MOLECULAR BASIS OF AN EVOLVED STRATEGY

Despite the prevalence of G protein-coupled receptors as transducers of signals from hormones, neurotransmitters, odorants, and light, little is known about mechanisms that regulate their plasma membrane expression (PME), although misfolded receptors are recognized and retained by a cellular quality control system (QCS). Convergent evolution of the gonadotropin-releasing hormone (GnRH) receptor (GnRHR) progressively decreases inositol phosphate production in response to agonist, validated as a measure of PME of receptor. A pharmacological chaperone that optimizes folding also increases PME of human, but not of rat or mouse, GnRHR because a higher percentage of human GnRHRs are misfolded structures due to their failure to form an apparent sulfhydryl bridge, and they are retained by the QCS. Bridge formation is increased by deleting (primate-specific) Lys191. In rat or mouse GnRHR that lacks Lys191, the bridge is non-essential and receptor is efficiently routed to the plasma membrane. Addition of Lys191 alone to the rat sequence did not diminish PME, indicating that other changes are required for its effects. A strategy, based on identification of amino acids that both 1) co-evolved with the Lys191 and 2) were thermodynamically unfavorable substitutions, identified motifs in multiple domains of the human receptor that control the destabilizing influence of Lys191 on a particular Cys bridge, resulting in diminished PME. The data show a novel and underappreciated means of posttranslational control of a G protein-coupled receptor by altering its interaction with the QCS and provide a biochemical explanation of the basis of disease-causing mutations of this receptor.