Colleen A. Flanagan

Cloning and characterization of the human GnRH receptor

A cDNA encoding the human GnRH receptor (GnRHR) has been cloned and functionally expressed in both Xenopus oocytes and COS-1 cells. The 2160 bp cDNA encodes a 328 amino acid protein with a predicted amino acid sequence that is 90% identical to that of the mouse GnRHR (Tsutsumi et al. (1992) Mol. Endocrinol. 6, 1163-1169). Injection of synthetic RNA transcript into oocytes led to the development of a depolarizing response to agonists when assayed by voltage-clamp electrophysiology. Consistent with the expression of a mammalian GnRHR, the response was blocked by GnRH antagonists. Following expression of the human GnRHR in COS-1 cells, agonists and an antagonist displaced [125I]GnRH agonist from membrane isolates with nanomolar range dissociation constants similar to those described for displacement from human pituitary membranes. Transfected COS-1 cells manifested a GnRH-stimulated increase in phosphoinositol turnover, with an EC50 of approximately 3 nM, which was inhibited by GnRH antagonists. Northern blot analysis revealed a single band of approximately 4.7 kb expressed in human pituitary which was not detected in testis. The predicted structure of the human GnRHR is similar to that previously reported for the mouse receptor. Although the mammalian GnRHR is a seven transmembrane domain receptor, it differs from other G-protein coupled receptors in several respects, most notably the lack of a cytoplasmic C-terminal domain. The present study demonstrates that the cDNA isolated encodes the human GnRHR and suggests that several unique features conserved among mammalian GnRHRs may be essential for receptor function and/or regulatory control.

Identification of N-glycosylation sites in the gonadotropin-releasing hormone receptor: role in receptor expression but not ligand binding

The asparagine residues of the three N-glycosylation consensus sequences in the mouse gonadotropin-releasing hormone receptor were mutated to determine which residues were glycosylated and the function of glycosylation. Photoaffinity labelled Gln4 and Gln18 receptor mutants exhibited lower apparent molecular weight on SDS polyacrylamide gel electrophoresis, while the Gln102 receptor showed wildtype mobility. This indicates that the receptor is glycosylated at Asn4 and Asn18 but not at Asn102. Binding affinities of all the mutant receptors were normal, indicating that carbohydrate moieties are not involved in ligand binding interactions. However, expression of the Gln4 and Gln18 receptors were substantially decreased, indicating a role for glycosylation in receptor expression or stability. All the glycosylation site mutants were capable of normal signal transduction, as indicated by their ability to stimulate inositol phosphate production.

The Functional Microdomain in Transmembrane Helices 2 and 7 Regulates Expression, Activation, and Coupling Pathways of the Gonadotropin-releasing Hormone Receptor

Structural microdomains of G protein-coupled receptors (GPCRs) consist of spatially related side chains that mediate discrete functions. The conserved helix 2/helix 7 microdomain was identified because the gonadotropin-releasing hormone (GnRH) receptor appears to have interchanged the Asp2.50 and Asn7.49 residues which are conserved in transmembrane helices 2 and 7 of rhodopsin-like GPCRs. We now demonstrate that different side chains of this microdomain contribute specifically to receptor expression, heterotrimeric G protein-, and small G protein-mediated signaling. An Asn residue is required in position 2.50(87) for expression of the GnRH receptor at the cell surface, most likely through an interaction with the conserved Asn1.50(53) residue, which we also find is required for receptor expression. Most GPCRs require an Asp side chain at either the helix 2 or helix 7 locus of the microdomain for coupling to heterotrimeric G proteins, but the GnRH receptor has transferred the requirement for an acidic residue from helix 2 to 7. However, the presence of Asp at the helix 7 locus precludes small G protein-dependent coupling to phospholipase D. These results implicate specific components of the helix 2/helix 7 microdomain in receptor expression and in determining the ability of the receptor to adopt distinct activated conformations that are optimal for interaction with heterotrimeric and small G proteins.

A Chicken Gonadotropin-releasing Hormone Receptor That Confers Agonist Activity to Mammalian Antagonists IDENTIFICATION OF d-LYS6 IN THE LIGAND AND EXTRACELLULAR LOOP TWO OF THE RECEPTOR AS DETERMINANTS

Mammalian receptors for gonadotropin-releasing hormone (GnRH) have over 85% sequence homology and similar ligand selectivity. Biological studies indicated that the chicken GnRH receptor has a distinct pharmacology, and certain antagonists of mammalian GnRH receptors function as agonists. To explore the structural determinants of this, we have cloned a chicken pituitary GnRH receptor and demonstrated that it has marked differences in primary amino acid sequence (59% homology) and in its interactions with GnRH analogs. The chicken GnRH receptor had high affinity for mammalian GnRH (K i 4.1 ± 1.2 nm) , similar to the human receptor (K i 4.8 ± 1.2 nm). But, in contrast to the human receptor, it also had high affinity for chicken GnRH ([Gln8]GnRH) and GnRH II ([His5,Trp7,Tyr8]GnRH) (K i 5.3 ± 0.5 and 0.6 ± 0.01 nm). Three mammalian receptor antagonists were also pure antagonists in the chicken GnRH receptor. Another three, characterized by d-Lys6 ord-isopropyl-Lys6 moieties, functioned as pure antagonists in the human receptor but were full or partial agonists in the chicken receptor. This suggests that the Lys side chain interacts with functional groups of the chicken GnRH receptor to stabilize it in the active conformation and that these groups are not available in the activated human GnRH receptor. Substitution of the human receptor extracellular loop two with the chicken extracellular loop two identified this domain as capable of conferring agonist activity to mammalian antagonists. Although functioning of antagonists as agonists has been shown to be species-dependent for several GPCRs, the dependence of this on an extracellular domain has not been described.

[8] Ligand binding and second-messenger assays for cloned Gq/G11-coupled neuropeptide receptors: The GnRH receptor

Publisher Summary This chapter discusses ligand binding and second-messenger assays for cloned Gq/G11-coupled neuropeptide receptors. The identification and characterization of cloned G protein-coupled neuropeptide receptors require methodology for monitoring ligand binding and coupling to intracellular signaling pathways in transfected cells. The availability of both monitoring systems is a prerequisite for mutagenesis studies directed at the molecular dissection of ligand-binding sites, molecular switches mediating agonist activation of the receptor, and domains involved in G protein binding and activation for signal transduction. Neuropeptidergic neurons and their targets in the central and peripheral nervous systems represent a major regulatory component, which is even more prevalent than classical biogenic amine neurons and their targets. Neuropeptides also encompass a far more diverse array of molecular signaling structures, whose importance in the regulation of neural and endocrine pathways is reflected in the increasing quest for neuropeptide analogs by the pharmaceutical industry. The essential components of a neuropeptide receptor assay are a high specific activity and high-affinity radiolabeled ligand, a relatively rich source of receptor (membrane-associated or solubilized), and a method for separating bound and free ligand.

Incorporation of an additional glycosylation site enhances expression of functional human gonadotropin-releasing hormone receptor

Mutation ofN-glycosylation sites in the mouse gonadotropin-releasing hormone receptor was previously shown to impair its expression in COS-1 cells. We therefore investigated the effects of adding an extra glycosylation site to the human gonadotropin-releasing hormone receptor, as a means for increasing its expression. Covalent labeling of the mutant receptor expressed in COS-1 cells with a gonadotropin-releasing hormone (GnRH) photoreactive analog demonstrated a shift in apparent molecular weight, indicating that the new site was in fact glycosylated. The receptor with extra glycosylation site displayed normal binding affinities for agonists buserelin and [d-Ala6-Pro9-NHEt]-GnRH, and the antagonist antide, and a slightly increased affinity for GnRH. Receptor number was increased by 1.7-fold in membrane preparations from cells expressing the mutant receptor, compared with wild-type. Photoaffinity labeling of cell-surface receptors in intact cells demonstrated a 1.8-fold increase in binding sites on the cell surface. The GnRH receptor (GnRHR) with extra glycosylation site conferred a markedly enhanced signaling response to agonist. Dose-response curves for GnRH-stimulated inositol phosphate production were left-shifted by an average of 4.4-fold, and maximal inositol phosphate responses were increased by 1.2 fold, in cells transfected with mutant compared with wild-type receptor, indicating that the increase in binding sites represented functional receptors. These results demonstrate that addition of an extra glycosylation site enhances expression of the human GnRHR, a strategy that may be applicable to other cell-surface receptors.

A Crucial Role for Gαq/11, But Not Gαi/o or Gαs, in Gonadotropin-Releasing Hormone Receptor-Mediated Cell Growth Inhibition

GnRH acts on its cognate receptor in pituitary gonadotropes to regulate the biosynthesis and secretion of gonadotropins. It may also have direct extrapituitary actions, including inhibition of cell growth in reproductive malignancies, in which GnRH activation of the MAPK cascades is thought to play a pivotal role. In extrapituitary tissues, GnRH receptor signaling has been postulated to involve coupling of the receptor to different G proteins. We examined the ability of the GnRH receptor to couple directly to Galpha(q/11), Galpha(i/o), and Galpha(s), their roles in the activation of the MAPK cascades, and the subsequent cellular effects. We show that in Galpha(q/11)-negative cells stably expressing the GnRH receptor, GnRH did not induce activation of ERK, jun-N-terminal kinase, or P38 MAPK. In contrast to Galpha(i) or chimeric Galpha(qi5), transfection of Galpha(q) cDNA enabled GnRH to induce phosphorylation of ERK, jun-N-terminal kinase, and P38. Furthermore, no GnRH-mediated cAMP response or inhibition of isoproterenol-induced cAMP accumulation was observed. In another cellular background, [35S]GTPgammaS binding assays confirmed that the GnRH receptor was unable to directly couple to Galpha(i) but could directly interact with Galpha(q/11). Interestingly, GnRH stimulated a marked reduction in cell growth only in cells expressing Galpha(q), and this inhibition could be significantly rescued by blocking ERK activation. We therefore provide direct evidence, in multiple cellular backgrounds, that coupling of the GnRH receptor to Galpha(q/11), but not to Galpha(i/o) or Galpha(s), and consequent activation of ERK plays a crucial role in GnRH-mediated cell death.

Identification of Tyr290(6.58) of the Human Gonadotropin-Releasing Hormone (GnRH) Receptor as a Contact Residue for Both GnRH I and GnRH II: Importance for High-Affinity Binding and Receptor Activation†

Molecular modeling showed interactions of Tyr (290(6.58)) in transmembrane domain 6 of the GnRH receptor with Tyr (5) of GnRH I, and His (5) of GnRH II. The wild-type receptor exhibited high affinity for [Phe (5)]GnRH I and [Tyr (5)]GnRH II, but 127- and 177-fold decreased affinity for [Ala (5)]GnRH I and [Ala (5)]GnRH II, indicating that the aromatic ring in position 5 is crucial for receptor binding. The receptor mutation Y290F decreased affinity for GnRH I, [Phe (5)]GnRH I, GnRH II and [Tyr (5)]GnRH II, while Y290A and Y290L caused larger decreases, suggesting that both the para-OH and aromatic ring of Tyr (290(6.58)) are important for binding of ligands with aromatic residues in position 5. Mutating Tyr (290(6.58)) to Gln increased affinity for Tyr (5)-containing GnRH analogues 3-12-fold compared with the Y290A and Y290L mutants, suggesting a hydrogen-bond between Gln of the Y290Q mutant and Tyr (5) of GnRH analogues. All mutations had small effects on affinity of GnRH analogues that lack an aromatic residue in position 5. These results support direct interactions of the Tyr (290(6.58)) side chain with Tyr (5) of GnRH I and His (5) of GnRH II. Tyr (290(6.58)) mutations, except for Y290F, caused larger decreases in GnRH potency than affinity, indicating that an aromatic ring is important for the agonist-induced receptor conformational switch.

Pro7.33(303) of the human GnRH receptor regulates selective binding of mammalian GnRH.

Mammalian gonadotropin releasing hormone (GnRH) receptors have a conserved acidic residue (Glu7.32(301) or Asp7.32(302)) in extracellular loop (ECL) three that confers selectivity for mammalian GnRH, which has Arg8. Comparison of mammalian and non-mammalian GnRH receptors suggested that the acidic residue is not the only determinant of ligand selectivity in mammalian receptors. The acidic residue is followed by a conserved Pro7.33 in mammalian GnRH receptors, but not non-mammalian receptors. Unique structural constraints imposed by Pro residues suggested that Pro7.33 determines selective binding of Arg8-containing GnRH, by stabilising the conformation of the third extracellular loop of the receptor. Substituting Pro7.33(303) or introducing Pro to position 7.31 decreased affinity for GnRH, but not analogs lacking Arg8. Substituting Pro7.33(303) changed the predicted alpha-helix content of the loop-helix interface. These results show that Pro7.33(303) of the human GnRH receptor is required for selective high affinity binding of mammalian GnRH and supports the hypothesis that Pro7.33(303) stabilises a loop conformation that is necessary for selective ligand binding.

Agonist activity of mammalian gonadotropin-releasing antagonists in chicken gonadotropes reflects marked differences in vertebrate gonadotropin-releasing receptors

The pharmacology of mammalian and avian gonadotropin-releasing (GnRH) receptors differs for agonist analogues. We have therefore compared the activities of mammalian-based GnRH antagonists in sheep and chicken gonadotropes to further elucidate the different structural requirements of the receptors. The antagonist activities of ten GnRH analogues were compared in cultured sheep and chicken pituitary cells by determining the dose required to cause a 50% inhibition of luteinizing hormone secretion (IC50) induced by GnRH at its half-maximal concentration (EC50). Nine analogues showed high antagonist activity in the sheep bioassay. Analogue IC50s varied between half and twice ((1.22-6.06) x 10(-10) M) the GnRH EC50 (3 x 10(-10) M). One of these peptides exhibited partial agonist activity. In contrast, eight of the analogues showed low antagonist activity in chicken pituitary cells, with IC50s varying from 46 to 1490 times ((1.4-44.7) x 10(-7) M) the GnRH EC50 (3 x 10(-9) M) and had a different order of potencies compared with that in the sheep. Furthermore, two analogues did not display antagonist activity at all in the chicken bioassay, but acted as pure agonists, stimulating LH secretion. These findings demonstrate marked differences in pharmacology between the avian and mammalian pituitary GnRH receptors and emphasize that GnRH antagonists, selected for their efficacy in mammals, cannot necessarily be used for physiological studies in non-mammalian vertebrates. The distinctly different pharmacology of the receptors and structural requirements of analogues for agonist/antagonist activity establish a basis for identifying receptor features involved in ligand-induced signal propagation using chimaeras of cloned sheep and chicken receptors.