Gonadotropin-Releasing Hormone Receptors

Gonadotropin-Releasing Hormone Receptors

ROBERT P. MILLAR, ZHI-LIANG LU, ADAM J. PAWSON, COLLEEN A. FLANAGAN,KEVIN MORGAN, AND STUART R. MAUDSLEY

Medical Research Council Human Reproductive Sciences Unit (R.P.M., Z.-L.L., A.J.P., K.M., S.R.M.), Centre forReproductive Biology, Edinburgh EH16 4SB, Scotland, United Kingdom; and Division of Medical Biochemistry andDepartment of Medicine (R.P.M., C.A.F.), University of Cape Town Faculty of Health Sciences, Cape Town 7925,South Africa

GnRH and its analogs are used extensively for the treatment ofhormone-dependent diseases and assisted reproductive tech-niques. They also have potential as novel contraceptives in menand women. A thorough delineation of the molecular mecha-nisms involved in ligand binding, receptor activation, and in-tracellular signal transduction is kernel to understanding dis-ease processes and the development of specific interventions.Twenty-three structural variants of GnRH have been identifiedin protochordates and vertebrates. In many vertebrates, threeGnRHs and three cognate receptors have been identified withdistinct distributions and functions. In man, the hypothalamicGnRH regulates gonadotropin secretion through the pituitaryGnRH type I receptor via activation of Gq. In-depth studies haveidentified amino acid residues in both the ligand and recep-tor involved in binding, receptor activation, and translation intointracellular signal transduction. Although the predominantcoupling of the type I GnRH receptor in the gonadotrope is

through productive Gq stimulation, signal transduction can oc-cur via other G proteins and potentially by G protein-indepen-dent means. The eventual selection of intracellular signalingmay be specifically directed by variations in ligand structure. Asecond form of GnRH, GnRH II, conserved in all higher verte-brates, including man, is present in extrahypothalamic brainand many reproductive tissues. Its cognate receptor has beencloned from various vertebrate species, including New and OldWorld primates. The human gene homolog of this receptor, how-ever, has a frame-shift and stop codon, and it appears that GnRHII signaling occurs through the type I GnRH receptor. There hasbeen considerable plasticity in the use of different GnRHs, re-ceptors, and signaling pathways for diverse functions. Delinea-tion of the structural elements in GnRH and the receptor, whichfacilitate differential signaling, will contribute to the develop-ment of novel interventive GnRH analogs. (Endocrine Reviews25: 235–275, 2004)

I. IntroductionII. Structure of GnRHs and Analogs

A. Structural variants of GnRHsB. Structure of GnRH and peptide analogsC. The evolutionarily conserved GnRH IID. Nonpeptide GnRH antagonists

III. Structure of GnRH ReceptorsA. Primary structures of GnRH receptorsB. Tertiary structure of the mammalian type I GnRH

receptorIV. Binding of GnRH to the Mammalian Type I GnRH

ReceptorA. Aspartate2.61(98) [D2.61(98)]B. Asparagine2.65(102) [N2.65(102)]C. Lysine3.32(121) [K3.32(121)]D. Asparagine5.39(212) [N5.39(212)]E. Tyrosine6.58(290) [Y6.58(290)]F. Aspartate7.32(302) [D7.32(302)]G. Effects of mutations of other residues on the ligand

binding pocketH. Ligand docking to the receptor

V. Binding Interactions of Other GnRH Ligands and OtherReceptors

A. GnRH IIB. Peptide agonistsC. Peptide antagonistsD. Nonpeptide antagonistsE. Binding sites in nonmammalian type I GnRH receptorsF. Binding sites in type II receptorsG. Utilization of binding sites common to the rhodopsin

family of GPCRsVI. Receptor Activation

A. Interaction of Asn2.50(87)/Asp7.49(319) in TM 2/7 inGnRH receptor activation

B. Disruption of TM3 Asp3.49(138)/Arg3.50(139) interactionin GnRH receptor activation

C. The triad of Glu2.53(90)-Lys3.32(121)-Asp2.61(98)

D. Role of extracellular loop 2E. Other residues possibly involved in receptor activationF. Integrated model of GnRH receptor activation

VII. GnRH Receptor Mutations in HypogonadotropicHypogonadism

VIII. Structural Correlates of GnRH Receptor Coupling andInternalizationA. Coupling to multiple G proteinsB. Regulators of G protein signaling (RGS) proteinsC. GnRH receptor internalization

IX. Conclusions and Future Perspectives

I. Introduction

GnRH IS THE central regulator of the reproductive hor-monal cascade and was first isolated from mamma-

lian hypothalami as the decapeptide (pGlu-His-Trp-Ser-Tyr-

Abbreviations: EC, Extracellular loop; GPCR, G protein-coupled re-ceptor; 5HT2A, 5-hydroxytryptamine (2A); IC, intracellular loop; NMR,nuclear magnetic resonance; PLC, phospholipase C; PLD, phospholipaseD; RGS, regulator of G protein signaling; TM, transmembrane.Endocrine Reviews is published bimonthly by The Endocrine Society(http://www.endo-society.org), the foremost professional society serv-ing the endocrine community.

0163-769X/04/$20.00/0 Endocrine Reviews 25(2):235–275Printed in U.S.A. Copyright © 2004 by The Endocrine Society

doi: 10.1210/er.2003-0002

235

Gly-Leu-Arg-Pro-Gly.NH2) (1–3). GnRH is processed inhypothalamic neurons from a precursor polypeptide by en-zymic processing and packaged in storage granules that aretransported down axons to the external zone of the medianeminence (4, 5). The peptide is released in synchronizedpulses from the nerve endings of about 1000 neurons into thehypophyseal portal system every 30–120 min to stimulate thebiosynthesis and secretion of LH and FSH from pituitarygonadotropes (4). Each GnRH pulse stimulates a pulse of LHrelease, but FSH pulses are less distinct. Although LH isstored and largely dependent on GnRH for secretion, FSHtends to be constitutively secreted and is more dependent onbiosynthesis for its secretion. The frequency of pulses is high-est at the ovulatory LH surge and lowest during the lutealphase of the ovarian cycle. The asynchronous patterns of LHand FSH release result from changes in GnRH pulse fre-quency, modulating effects of gonadal steroid and peptidehormones on FSH and LH responses to GnRH, and differ-ences in the half-lives of the two hormones.

Low doses of synthetic GnRH delivered in a pulsatilefashion to simulate the endogenous GnRH levels in the portalvessels (picograms per milliliter) restore fertility in hypogo-nadal men and women and are also effective in the treatmentof undescended testes and delayed puberty (6–12). How-ever, high doses of GnRH or agonist analogs desensitize thegonadotrope with resultant decrease in LH and FSH and adecline in ovarian and testicular function (6–13). This de-sensitization phenomenon is extensively applied in clinicalmedicine for the treatment of a wide range of diseases (6–13)(Table 1). GnRH peptide antagonists also inhibit the repro-ductive system through competition with endogenousGnRH for receptor binding, but the doses required are higherthan the desensitizing agonist doses, presenting challengesfor administration in the treatment of chronic diseases (14).The development of novel delivery systems for peptide an-tagonists or the development of nonpeptide orally activeGnRH antagonists (14) is therefore likely to replace agonisttherapy and avoid the undesirable stimulation and diseaseflare that precedes desensitization. In addition to the thera-peutic applications, GnRH analogs are predicted to be used

as new generation male and female contraceptives in con-junction with steroid hormone replacement (15–17).

The extensive clinical applications of GnRH analogs haveattracted detailed studies of the physiology, cell biology, andmolecular function of the hormone. These studies offer thepotential to enhance our understanding of the entire systemand for the optimal application of analog therapies. Themolecular cloning of GnRH receptors accelerated progress instudies of the structure-activity of the receptor-ligand com-plex (18–21). The advances in contextualizing the knownstructure-activity relations of GnRH and its analogs withtheir interactions with the GnRH receptor were extensivelyreviewed (18). During the ensuing 5 yr, there has been furtherprogress resulting from additional receptor mutagenesisstudies, the solving of the structure of rhodopsin to refineGnRH receptor molecular models, the cloning of novelGnRH receptors, and the development of nonpeptide smallmolecule GnRH antagonists (14).

This article reviews our current knowledge on the struc-ture, ligand interactions, and activation of the type I GnRHreceptor. In addition, the array of novel GnRH receptors inmammals and nonmammals and their relationships and dif-ferences will be described. The review will only briefly covercurrent knowledge on GnRH structural variants, their pos-sible functions, and structure-activity relations of GnRH an-alogs because these have been thoroughly reviewed previ-ously and have not been subject to major new developments(18, 22–24). Nevertheless, information on the structures andsubtype classification of naturally occurring GnRHs andGnRH analogs is provided because this is required for thediscussion of ligand selectivity and ligand interactions ofGnRH receptors. Considerable attention will be given to themolecular functioning of the GnRH receptor as new insightshave recently emerged. The important area of GnRH-medi-ated intracellular signaling will not be covered because it hasbeen the subject of recent comprehensive review (25–30), butreceptor structural elements involved in binding and acti-vation of signaling proteins and internalization of receptorswill be addressed in detail.

TABLE 1. Clinical applications of GnRH and GnRH analogs

Pulsatile GnRH (stimulation)Infertility Stimulates gamete and hormone productionCryptorchidism Descent of testesDelayed puberty Advances puberty

GnRH agonists and antagonists (inhibition)Contraception Inhibition of ovulation and spermatogenesis with add-back sex

steroid hormonesHormone-dependent diseases Prostatic cancer

Benign prostatic hypertrophyBreast cancerEndometriosisUterine fibroidsPremenstrual syndromePolycystic ovarian syndromeHirsutismAcne vulgarisPrecocious pubertyAcute intermittent porphyria

Infertility Inhibition of endogenous gonadotropin together with controlledadministration of exogenous gonadotropin, especially ininduction of ovulation for assisted reproduction techniques

236 Endocrine Reviews, April 2004, 25(2):235–275 Millar et al. • GnRH Receptors

II. Structure of GnRHs and Analogs

A. Structural variants of GnRHs

Although mammalian GnRH isolated from the hypo-thalamus was thought to be a unique structure with aprimary role in regulating LH and FSH, it became appar-ent that diverse forms exist in vertebrates (31, 32). This hasled to the structural identification of 23 different forms(20 –24, 33– 42a) (Fig. 1). These are distributed in a widerange of tissues in vertebrates in which they apparently havediverse functions, including neuroendocrine (e.g., GH releasein certain fish species), paracrine (e.g., in placenta and go-nads), autocrine (e.g., GnRH neurons, immune cells, breast

and prostatic cancer cells), and neurotransmitter/neuro-modulatory roles in the central and peripheral nervous sys-tems (e.g., sympathetic ganglion, mid-brain) (6, 13, 18, 22–24,33–38, 43–45). Because none of this signaler/target cell com-munication is mediated through secretion of GnRH into thegeneral circulation, a single form of GnRH is theoreticallycapable of serving all of these roles (see Type II GnRH re-ceptor, Section III.A). However, it is evident that at least two,and usually three, forms of GnRH are present in the majorityof the vertebrate species studied (18, 20–24, 33–38). The mostubiquitous is chicken GnRH II, which was first isolated fromchicken brain (46). Because the chicken GnRH II structure istotally conserved from bony fish to man, this is probably the

FIG. 1. Primary amino acid sequences of naturally occurring GnRH structural variants spanning approximately 600 million yr of evolution.The shaded regions show the conserved NH2- and COOH-terminal residues that play important functional roles. Nonconserved residues areeither unimportant or convey ligand selectivity for a particular GnRH receptor. Note that the GnRHs are named according to the species inwhich they were first discovered, and they may be represented in more than one species. For example, mammalian GnRH is widely presentin amphibians and primitive bony fish. Chicken GnRH II (Chicken II) is present in most vertebrate species, including man and salmon. GnRH(GnRH III) is probably present in all teleost fish. For reviews, refer to Refs. 20–24 and 33–38. More recent discoveries of novel GnRHs fromRana (40), medaka (120), the sea squirt, Ciona (AV893326, AV974399, and Ref. 42), and octopus (49) are also shown. The octopus has twoadditional amino acids shown as an insert for alignment purposes.

Millar et al. • GnRH Receptors Endocrine Reviews, April 2004, 25(2):235–275 237

earliest evolved form and has critical functions (see SectionII.C). This form has been designated GnRH II, whereas thehypothalamic form is designated type I (18, 47). In manyvertebrate species, a third conserved form of GnRH (salmonGnRH) is localized to the terminal nerve in the forebrain inteleost fish and is designated GnRH III (48). GnRH III exhibitscomplete sequence conservation but occurs only in teleosts,suggesting that the gene encoding this peptide originatedafter the divergence of teleosts from the vertebrate lineage.Interestingly, sockeye salmon possess two genes encodingGnRH III. Structural analysis of the genes encoding theGnRHs supports this general classification into three forms(48), and this conclusion is confirmed by a more extensivephylogenetic analysis (Fig. 2).

The NH2-terminal amino acids (pGlu-His-Trp-Ser) andCOOH-terminal amino acids (Pro-Gly.NH2) are conservedover about 600 million yr of chordate evolution, with theexception of two conservative Tyr substitutions (Fig. 1). Thetype I GnRHs exhibit considerable variation in positions 5, 7,and 8, which affect ligand selectivity (see Section II.B).

The protochordate (chordate ancestor) sea squirt (Ciona)gene is interesting in that three GnRH forms are encoded intandem within single genes. The most ancient of the GnRHsidentified is a homolog identified in the octopus (49). Thismolecule exhibits the characteristic pGlu and Pro9Gly10.NH2but has an additional two amino acids inserted in the middleregion of the molecule. Nevertheless, it is capable of stimu-lating LH release from quail pituitary cells (49).

B. Structure of GnRH and peptide analogs

The conservation of the length of the peptide (10 aminoacids) and the NH2 terminus (pGlu-His-Trp-Ser) and COOHterminus (Pro-Gly.NH2) (Fig. 1) indicates that these featuresare critically important for receptor binding and activation.This is borne out from structure-activity data from severalthousand analogs that were developed largely on an empir-ical basis. Indeed, cognizance of the evolutionary constraintson GnRH structure identify the functionally important res-idues and would have obviated a considerable degree of theendeavor to produce agonists and antagonists. The consid-erable variation in position 8 of natural GnRHs (Arg, Gln,Trp, Ser, Thr, Asn, Leu, Tyr, Lys, Ala, Trp) suggests thatvirtually any residue is tolerated in this position. However,this is clearly not the case for the mammalian pituitary typeI GnRH receptor (18, 50), which requires Arg in position 8 forhigh-affinity binding. Recent work on cloned nonmamma-lian receptors also indicates certain specificities for the aminoacid in this position (35, 51–53). Thus, the residue in position8 seems to play an important role in ligand-selectivity of thedifferent GnRH receptors. The roles of the individual aminoacids comprising GnRH and the structure-activity relationsof agonist and antagonist analogs have been extensively re-viewed (18, 50) and will not be repeated here.

Short peptides such as GnRH are highly flexible in solutionand exist as an equilibrium between numerous conforma-tions (54–57). However, among these conformations are so-called bioactive conformations that represent preferredstructures for interaction with the receptor. For GnRH, thebioactive conformation is the product of a number of influ-

ences, which include intramolecular interactions, local in-fluences of solvents (water), lipids, and initial receptor in-teractions that conform the ligand. In addition, membraneand intracellular proteins associate with the receptor andalter its conformation and selectivity for ligand conforma-tions. Studies on GnRH and its receptor are increasinglypointing to a multiplicity of bioactive conformations of boththe ligand and receptor. These in turn result in differentialactivation of intracellular signaling pathways (our unpub-lished observations).

The first studies on GnRH structure by conformationalenergy analysis of the NH2-terminal 1–6 and carboxyl-ter-minal 6–10 amino acids identified a low energy CC con-former that featured a �-II’ type turn involving Tyr5-Gly6-Leu7-Arg8 such that the NH2 and COOH termini are closelyapposed (55) (Fig. 3). This conclusion has subsequently beensupported by a variety of experimental data with syntheticGnRH analogs (58–61), interactions with region-specific an-tibodies (62), and a range of physicochemical studies. Thesestudies have been extensively reviewed (18, 50) and will onlybe briefly covered here. A recent study using electron capturedissociation mass spectrometry has confirmed the presenceof the �-II’ type turn and interaction of the NH2 and COOHtermini (N. C. Polfer, personal communication).

The �-II’ type turn involving residues 5–8 is partly due tointramolecular interactions with the side chain of Arg8, asvarious studies, including Trp fluorescence (63, 64), com-puter simulations using the technique of conformationalmemories (57), and nuclear magnetic resonance (NMR) (54)have shown that substitution of Arg8 (e.g., with Gln8 as inchicken GnRH I) results in a more extended structure witha loss of predominance of the folded conformers and a lowbiological activity (Fig. 4). Yet these extended forms (e.g.,Gln8GnRH) have high activity in many nonmammalianGnRH receptors (18, 51, 52, 65–67) despite their low activityat the mammalian receptor (18). The �-II’ type turn confor-mation of GnRH also appears to be induced in part by theinteraction of Arg8 with an acidic residue [Asp7.32(302)] inextracellular loop (EC) 3 of the mammalian receptor (18,68–70) (Fig. 5). Substitution of a d-amino acid for Gly6 ap-parently enhances the �-II’ type turn conformation and in-creases the activity of Arg8 GnRH about one to two ordersof magnitude at mammalian receptors (18, 50). The d-aminoacid substitution overcomes the deleterious effects of Arg8

substitution (e.g., with Gln8) such that binding affinity for themammalian receptor is increased almost 1000-fold (52, 68, 69)(see Section V.A).

Although nonmammalian GnRHs appear to interact withtheir cognate receptors through predominantly the samebinding sites as those of in the mammalian GnRH receptor,they appear not to require the �-II’ folded conformation ofthe ligand because these GnRHs (e.g., chicken GnRH I,Gln8GnRH) are less configured and more extended in theirstructure (54, 57). This suggests that substitution of a d-aminoacid for Gly6 would not enhance binding affinity for non-mammalian receptors (18, 23, 24). Although limited exper-imental data were presented in support of this (22, 71), moreextensive studies with a wider range of analogs demon-strated that d-amino acid substitution of Gly6 in mammalian


and nonmammalian GnRHs did enhance binding affinity atthe chicken, catfish, bull frog, and Xenopus receptors (72).

The amino-terminal residues of GnRH are involved inreceptor activation, and modification of these residues inGnRH produces analogs with antagonistic properties (18, 50)(Figs. 3 and 5). As in agonists, substitution of Gly6 with ad-amino acid enhances the activity of the antagonists. Be-

cause the antagonists have high binding affinity, the loss ofamino-terminal contacts in agonists that activate the receptoris presumably compensated for by new contacts made by thesubstituted amino acids in antagonists.

The first generation of potent GnRH antagonists werecharacterized by high histamine-releasing properties as aresult of the presence of basic residues (basic-X-basic se-

FIG. 2. Unrooted phylogenetic tree constructed from primary amino acid sequences of cloned GnRH ligand precursors in the Genbank database.Clustal alignments were generated using GeneJockey II software (Biosoft UK, Cambridge, UK), and phylogenetic trees were generated usinga topological algorithm with PHYLIP software available at the Russian EMBnet Node: http://www.genebee.msu.su/emb.html. Bootstrap valuesare not indicated, and branch lengths are approximated. Identification of the source of the genes is beyond the scope of this review and willappear elsewhere.


quence). Elimination of basicity produced analogs withlower histaminic properties but poorer solubilities and thetendency to form gels. This has resulted in difficulties informulation that continue to be a problem in GnRH antag-onists that are currently under clinical investigation. Theprimary structures of GnRH analogs that are extensivelyemployed therapeutically, or are in clinical development, areshown in Fig. 5.

GnRH II is an intriguing exception to the general conclu-sion that a d-amino acid substitution for Gly6 enhances thebinding affinity of GnRHs (72) (Table 2). An explanation maybe that GnRH II is already stabilized in the �-II’ turn con-formation and that incorporation of a d-amino acid in po-sition 6 does not further stabilize this conformation. ResiduesHis5, Trp7, and Tyr8 are proposed to contribute to the sta-bilization of GnRH II (72). Gly6 is essential to allow assump-

FIG. 3. Schematic representation of mammalian GnRH in the folded conformation in which it is bound to the GnRH pituitary receptor. Themolecule is bent around the flexible glycine in position 6. Substitution with D-amino acids in this position stabilizes the folded conformation,increases binding affinity, and decreases metabolic clearance. This feature is incorporated in all agonist and antagonist analogs (Fig. 5). TheNH2 (red) and COOH (green) termini are involved in receptor binding. The NH2 terminus alone is involved in receptor activation andsubstitutions in this region produce antagonists (see Fig. 5). [Adapted from R. P. Millar, Reproductive medicine: molecular cellular and geneticfundamentals (edited by B. C. J. M. Fauser), Parthenon Publishing, Lancaster, UK, 2002, pp 199–224 (21).]

FIG. 4. Schematic of NMR analysis of the structure of mammalian and chicken GnRH I (54). Mammalian GnRH exhibits three majorfamilies of conformers similar to the one shown in panel A. All three show a �-II’ turn about Gly6 and the NH2 and COOH termini in closeproximity. Although several hydrogen bonds were found in the three structures, only one between the carbonyl oxygen of Ser4 and theside chain amino group hydrogen of Arg8, and another between the Gly10.NH hydrogen and the pGlu1 carbonyl oxygen are present in allthree conformers. The Arg8 side chain is involved in at least one other hydrogen bond, with either the His2 side chain or the Tyr5 sidechain. The chicken GnRH I structures fall into four main families of conformers that differ from each other to a greater extent but areall extended forms as represented by panel B. None of the conformers have the hydrogen bonding of the mammalian GnRH, but a seriesof other hydrogen bonds of which a Ser4 bond to pGlu1 is the only one present in all four conformers. [Adapted from J. C. Maliekal et al.:S Afr J Chem 50:217–219, 1997 (54).]


tion of the folded conformation, and the NH2- and COOH-terminal sequences are essential for receptor binding andactivation. Thus, all the amino acids appear to have crucialroles, and this offers an explanation for the total conservationof GnRH II structure over 500 million yr of evolution.

The GnRHs in the primitive jawless lamprey, proto-chordates, and octopus lack the conserved Gly6 of theGnRHs of jawed vertebrates (Fig. 1). The presence of chiralamino acids in place of the achiral Gly prevents the �-II’turn, which results in low binding affinity at the mam-malian pituitary receptor (18, 50). This suggests that thereceptors in these lower organisms do not have a require-ment for a folded conformation of GnRH and that thisfeature first evolved in the receptors of the bony fish. Wehave recently found that replacement of Ala6 in Ciona I(Fig. 1) with Gly restores binding affinity at the humantype I GnRH receptor and that substitution with d-Alafurther enhances binding affinity (R. P. Millar, unpub-lished observations).

C. The evolutionarily conserved GnRH II

As mentioned earlier, a second form of GnRH identifiedfrom chicken brain (chicken GnRH II, GnRH II) (Fig. 1) isubiquitous in vertebrates from primitive bony fish to man(22–24, 33–38, 73, 74). This complete conservation of structureover 500 million yr suggests that GnRH II has an importantfunction and a discriminating receptor (or receptors) that hasselected against any structural change in the ligand. Thispoints to essential functions that have yet to be definitivelyidentified. The wide distribution of GnRH II in the centraland peripheral nervous systems suggests a neurotransmit-ter/neuromodulatory role. This has been thoroughly dem-onstrated in the inhibition of M currents in the bullfrogsympathetic ganglion, which sensitizes neurons to depolar-ization (75, 76). GnRH II was identified in amphibian sym-pathetic ganglia, and the receptors present are highly selec-tive for the peptide (77).

Because GnRH had been shown to have direct effects on

FIG. 5. GnRH agonist and antagonist analogs in clinical practice or in clinical development.


sexual arousal in rodents (78–80) and type II GnRH is lo-calized in brain areas associated with reproductive behavior,it was suggested that this may be a role for the peptide (22–24,33, 79, 80). GnRH II and a GnRH II analog were both foundto be potent stimulators of reproductive behavior in ringdoves (23, 24), song sparrows (81), and the musk shrew (82).Recently, the cognate receptor for GnRH II was cloned fromthe marmoset and found to be distributed in those areas ofprimate brain associated with reproductive behaviors (45,83). Infusion of GnRH II into the third ventricle of femalemarmosets increased sexual behavior (D. Abbott, personalcommunication).

In addition to its apparent role as a neuromodulator in thenervous system, GnRH II and its receptor are present inreproductive tissues (45). GnRH binding sites and antipro-liferative effects of GnRH analogs have also been describedin reproductive tissue tumors and their cell lines (see reviewsin Refs. 6, 8, 11, 13, and 45). Interestingly, GnRH and analogbinding, signaling, and pharmacological effects were notcharacteristic of classical hypophysial type I GnRH receptorsbut are more similar to type II GnRH receptors (13, 83).Although this suggests that the antitumor effects in humantissues are mediated via the type II GnRH receptors (84), thehuman type II receptor gene is disrupted by a frame-shift anda stop codon, and a transcript that could encode a full-lengthreceptor or the expressed receptor protein has not been iden-tified (85, 86, 86a). It has now become apparent that thedifferent pharmacology of GnRH analogs in affecting pitu-itary function and in inhibiting proliferation of tumor celllines can both be mediated by the human type I GnRHreceptor by coupling through different signaling pathways(viz. Gq for pituitary and Gi for tumor cells). This differentialcoupling can be accomplished through both ligand selectiv-ity and intracellular milieu and will be discussed later in thisreview (see Section VIII.A).

D. Nonpeptide GnRH antagonists

The development of nonpeptide GnRH antagonists hasseen intense endeavors from the pharmaceutical industry.Representative compounds are shown in Fig. 6. The firstdescribed nonpeptide GnRH antagonist (compound 1) is afused tetracyclic benzodiazepine that blocks ovulation in rats

when given at a dose of 0.5 mg/kg (87). The antifungal drugketoconazole (Nizoral, Janssen Pharmaceutica, Beerse, Bel-gium) (compound 2) was found to bind and inhibit the ratpituitary GnRH receptor with an apparent IC50 of 2 �m.Addition of a number of groups to this core structure, suchas dipeptides and tripeptides related to GnRH, improvedaffinity to approximately 500 nm (88).

The cloning and ectopic expression of the human GnRHreceptor made screening of small molecular compound col-lections possible and the identification of lead molecules thatbind the human receptor. This resulted in a series of patentsfrom Takeda Pharmaceuticals describing benzodiazepines(89) (compound 3), spiroamines (90) (compound 4), andthienopyridones (91, 92) (compound 5). Unlike peptide an-alogs, which have for the most part shown similar affinitiesfor a variety of mammalian species, these small moleculescan exhibit marked species selectivity as has been observedfor other neuropeptide receptors. For example, compound 4binds the rat receptor with high affinity (IC50 � 9 nm) butbinds the human receptor with much lower affinity (IC50 �400 nm). This trend was observed to a greater or lesser degreefor the entire series of analogs. Conversely, compounds such ascompound 5 are highly selective for human (IC50 � 0.2 nm)compared with the rat (60 nm). This low affinity for the ratreceptor can invalidate convenient and inexpensive in vivo as-says in laboratory rodents, thus hindering drug development.

Merck has described both indole (93) (compound 6) andquinolone-based (94–96) (compound 7) small molecule an-tagonists, and Abbott’s description of the ketoconazole wasfollowed by the discovery of an erythromycin A derivative(97) (compound 8). Takeda has reported a new series (92)(compound 9) based on compound 5, and Alanex Corp.developed compound 10 (98). The most recent reports are afurther series of derivatives of compound 6 by Merck (com-pound 11) with IC50 in subnanomolar concentrations andexcellent oral bioavailability (99) and a series of imidazol-pyrimid-5-ones from Neurocrine (compound 12) that bindthe human receptor in the low nanomolar range (100, 101).Recently, a new series of small molecule antagonists havebeen developed by Pfizer (102).

Although there are exceptions, the majority of the smallmolecule GnRH antagonists conform to a simple pharmaco-phore model. This comprises a requirement for a basic proto-natable nitrogen group (optionally substituted by lipophilicgroups), one or two aromatic groups, and an aliphatic lipophilicgroup arranged in a putative �-turn mimetic configuration (92).Some of the small molecule antagonists have progressed toclinical trial. Takeda’s thienopyrimidinedione (TAK-013) is inphase two for endometriosis and uterine fibroids, whereas theirthienopyridine-one (TAK-810) is in phase one. Neurocrine’spyrolopyrimidone (NBI-42902) is in phase one trials for a rangeof reproductive indications.

III. Structure of GnRH Receptors

A. Primary structures of GnRH receptors

The amino acid sequence of the GnRH receptor was firstdeduced for the mouse receptor cloned from the pituitary�T3 gonadotrope cell line (103). This sequence was con-

TABLE 2. Enhancementa of binding affinity by constraint with D-amino acid substitution of Gly6 or 6, 7 �-lactam bridge

Mouse receptor Chicken receptor Catfish receptor

mGnRHD-Trp6 74 8.6 14.0D-Ala6 7.3 3.4 7.1�-Lactam 5.4 7.3 5.6

cGnRH ID-Ala6 4.1 1.8 4.1�-Lactam 4.9 4.9 6.5

sGnRHD-Arg6 6.7 18.8 5.0

GnRH IID-Trp6 0.8 1.5 0.9D-Arg6 1.3 1.6 2.2

mGnRH, Mammalian GnRH; cGnRH, chicken GnRH; sGnRH,salmon GnRH.

a Binding affinity relative to the parent natural GnRHs.


FIG. 6. Examples of nonpeptide GnRH antagonists.


firmed (104), and it provided the basis for the cloning ofGnRH pituitary receptors from the rat (105–107), human(108, 109) (Fig. 7), sheep (110, 111), cow (112), and pig (113)that share over 80% amino acid identity. Homologs of themammalian GnRH receptors have also been cloned from amarsupial (possum) (114, 114a), catfish (65), two forms fromthe goldfish (51), bullfrog (67), brown frog (115), clawed toad(66), chicken (71), medaka (116), striped bass (117), trout(118), salmon (118a), cichlid (119), Japanese eel (120), am-berjack (CAB 65407), rubber eel (AD 49750), and seasquirt(120a). The nonmammalian receptors with greatest homol-ogy to the mammalian pituitary receptors have 42–47%amino acid identity with the mammalian receptors but 58–67% identity among each other. These are all designated astype I GnRH receptors (Figs. 8 and 9). It is not altogether clearfrom homology comparisons that the classification of themammalian and nonmammalian type I together is correct,but similarities in microdomains (e.g., EC3) support this.Because the evolutionary time separating amphibians andmammals is similar to that separating amphibians and bonyfish, the poor conservation of sequence of the mammaliantype I GnRH receptor with the nonmammalian receptorsimplies a sudden acceleration in evolutionary change in themammals. This may have been driven by the loss of thecarboxyl-terminal tail in the mammalian type I receptor,which is unique among G protein-coupled receptors(GPCRs). In the goldfish (51), zebra fish (47), catfish (121,122), and salmon (P. Swanson, unpublished observations),there are two isoforms (type Ia and type Ib) that have 70%amino acid identity. In the goldfish, they differ in type Ia

having a putative SH3 binding domain (poly proline se-quence) in the carboxyl-terminal tail, which potentially con-veys the possibility of coupling to MAPKs (S. R. Maudsley,unpublished observations).

The presence of three GnRH forms in most vertebratespecies suggested the existence of three cognate GnRH re-ceptor subtypes in an analogous manner to the human tachy-kinin receptor system. Because the EC3 domain is a majordeterminant of receptor selectivity for the GnRH structuralvariants, degenerate oligonucleotides to the conservedboundary transmembrane (TM) domains were used to am-plify this domain from genomic DNA from various verte-brates (47). This revealed novel type II receptor sequences.These sequences were then used to identify a human putativetype II GnRH receptor (45, 85, 123, 124) and then clonebullfrog (67), clawed toad (B. Troskie, unpublished obser-vations), marmoset (83), macaque, and green monkey (126)type II receptors (Fig. 8). The approach also allowed thecloning of type III GnRH receptors from the bullfrog (67). Thefindings along with the cloning of other GnRH receptorssuggest an early evolution of the three GnRH receptor sub-types in vertebrates which parallels that of the GnRH ligands(Fig. 9). As mentioned earlier (see Section II.C), we and othershave been unable to identify a human type II receptor tran-script lacking a frame-shift and internal stop codon. Thesetranscripts are therefore incapable of being translated to afull-length GPCR. This apparent silencing of the type II re-ceptor was very paradoxical given the extraordinary con-servation of the cognate GnRH II ligand from bony fish toman. Stop sites or deletions in similar positions are also

FIG. 7. Two-dimensional representation of the human GnRH receptor showing TM domains (boxed) connected by ECs and ICs. Putative ligandbinding sites (red) and residues thought to be important in receptor structure or binding pocket formation are shown in green letters. Theseinclude disulfide bond formation and glycosylation sites. Residues involved in receptor activation are shown in blue. Residues in squares arethe ones highly conserved throughout the rhodopsin family of GPCRs. Residues involved in coupling to G proteins are shown in orange. Proteinkinase C (PKC) and protein kinase A (PKA) phosphorylation sites are indicated.


FIG. 8. Clustal alignment of primary amino acid sequences of cloned vertebrate type I, II, and III GnRH receptors. The TM domains areboxed, and the ICs and ECs are indicated. The consensus for the most characteristic domain (EC3 going into TM7) of the three receptortypes is shown. This domain was used to clone the three receptor types. The amino acids are colored green for charged/polar, blue fornonpolar, red for nonpolar aromatic, and black for nonpolar sulfhydryl. Note that TM domains are predominantly hydrophobic, and loopdomains are hydrophilic. Figure continues on next page.


FIG. 8. Continued.


present in the chimpanzee, cow, and sheep (86a), whereas afully functional type II receptor is present in New and OldWorld monkeys and the pig, as well as amphibian and reptilespecies (45). The gene has been completely deleted in themouse and is apparently absent in fish (45). This intriguinglysporadic inactivation or deletion of the type II receptor genehas been reviewed and concluded to arise from plasticity inthe use of the GnRH receptor subtypes for signaling by thedifferent GnRHs (45). GnRH I, GnRH II (except in mouse),and the type I receptor have been universally conserved, incontrast to the silencing of the type II receptor in a numberof species. This has apparently arisen because the type Ireceptor is capable of binding the GnRH II with high affinitysuch that it can take over the role of the type II receptor,whereas the converse cannot occur due to the high ligandselectivity of the type II receptor for GnRH II. Two otherexplanations have been proposed for the frame-shift and thestop codon in the human type II receptor (45).

First, the frame-shift and stop codon are accommodated

posttranscriptionally and during translation. Numerousmechanisms of mRNA editing have been described andcould potentially repair the frame-shift and stop. In thisregard we have noted transcripts that splice out the stop.Alternatively, the stop codon can be translated as a seleno-cysteine by means of a specific tRNA and a selenocysteineinsertion sequence motif (127), which is present in the 3�untranslated region of the gene encoding human type IIreceptor. However, we were unable to demonstrate seleno-cysteine incorporation (86).

Second, a partial type II receptor is elaborated and is func-tional. A Kozac consensus start site follows the stop, andwhen this partial receptor sequence is expressed in COS cellsit down-regulates the expression of the type I receptor (A. J.Pawson, unpublished observations). Interestingly, the full-length cDNA (including frame-shift and stop) increases typeI expression. Could it be that the function of type II tran-scripts is to regulate type I expression and coupling? Theircoexpression in gonadotropes suggests that this is feasible.

FIG. 9. Unrooted phylogenetic tree of GnRH receptor sequence relationships generated using a topological algorithm with PHYLIP software(EMBnet). Bootstrap values are not indicated, and branch lengths are approximated. Identification of the source of the genes is beyond the scopeof this review and will appear elsewhere.


The stop codon has arisen independently in evolution in thesame vicinity and no other place in unrelated species. Thismight suggest that there is an advantage in having the stopcodon and producing partial receptor sequences. The pres-ence of type II receptors with or without the stop codon inclosely related species suggests that any advantage of thestop is only marginal.

GnRH receptor orthologs have been identified in Drosophilamelanogaster (128) and Caenorhabditis elegans (P. Swanson, un-published observations), indicating a very early evolutionaryorigin. However, the cognate ligand for the Drosophila GnRHreceptor homolog is not a GnRH, but is an adipokinetic hor-mone that has a similar structure in its length and the presenceof the NH2-terminal pGlu and a COOH-terminal amide (129).

We have examined the suggested classifications of GnRHreceptors by constructing phylogenetic trees. The primaryamino acid structures of cloned GnRH receptors werealigned and used to generate an unrooted tree using a to-pological algorithm that optimizes tree structure before de-termining branch lengths. This revealed that the receptorscan be grouped into distinct classes: types I, II, and III (Fig.9). Type I and type II GnRH receptors form elongated clus-ters. Type III GnRH receptors are more closely related to typeII receptors than to type I receptors, suggesting that type IIand type III receptors may have arisen from duplication ofan ancestral gene in lower vertebrates. The numerical nam-ing of individual cloned receptors in the Genbank databasefrequently does not comply with the phylogenetic related-ness because researchers have named them by pharmaco-logical characteristics, order of discovery, or tissue expres-sion (67, 115, 116, 122). For example, Bogerd et al. (122)recently named a novel catfish receptor R2, although it hasgreatest homology with goldfish 1a receptor (51). Similarly,Wang et al. (67) named the receptor cloned from bullfrogpituitary as bullfrog I, although its greatest homology is withtype III receptors. We have retained the original authors’designation in Fig. 9 to highlight these discrepancies. Clearly,a more systematic and consistent approach is required.

GnRH receptors have the characteristic features of GPCRs(Figs. 7 and 8). The NH2-terminal domain is followed byseven �-helical TM domains connected by three EC domainsand three intracellular loop (IC) domains. The extracellulardomains and superficial regions of the TMs are usually in-volved in binding of peptide hormones such as GnRH, andthe TMs are believed to be involved in receptor configurationand conformational change associated with signal propaga-tion (receptor activation). These changes are thought to prop-agate into conformational changes in the intracellular do-mains involved in interacting with G proteins and otherproteins for intracellular signal transduction.

A unique feature of the mammalian type I GnRH receptoris the absence of a carboxyl-terminal tail present in all otherGPCRs and in all of the nonmammalian and mammaliantype II GnRH receptors. This is, therefore, a recently evolvedfeature that presumably serves an important role in the func-tioning of the mammalian GnRH receptor (see Section VI.F).

The conservation of amino acids during evolution frombony fish to mammals is likely to identify those residues thatare crucial for GnRH receptor function (18, 35). These includeresidues thought to be involved in GnRH receptor binding

Asp2.61(98), Asn2.65(102), Lys3.32(121), Asn5.39(212), Tyr6.58(290), andAsp7.32(302) (Figs. 7 and 8). The conserved residues includethose conserved or conservatively substituted throughoutthe rhodopsin family of GPCRs. These are shown in Fig. 7 insquares and are the residues used as reference points for theconsensus numbering of the rhodopsin family of GPCRs [i.e.,Asn1.50(53), Asn2.50(87), Arg3.50(138), Trp4.50(164), Pro5.50(223),Pro6.50(282), and Pro7.50(320) (Ref. 18)] (see Section III.B).

B. Tertiary structure of the mammalian type IGnRH receptor

A knowledge of the three-dimensional structure of themammalian GnRH receptor is essential for an understandingof its molecular functioning. The only direct structural in-formation at atomic resolution of a GPCR is derived fromx-ray analysis of the ground state of rhodopsin (130). Pre-vious structural information on GPCRs was predicted fromlow resolution electron microscopy of bacteriorhodopsin andrhodopsin (131–133). Structural information for all otherGPCRs has relied on molecular models (18, 19, 134–138)based on the rhodopsin structure. A model (139) incorpo-rating structural information derived from the analyses ofapproximately 500 sequences in the rhodopsin-like family ofGPCRs ultimately turned out to be very similar to the struc-ture of rhodopsin, suggesting a similar structure of the sevenTM domains of all GPCRs in the rhodopsin family. In con-trast, the EC and IC domains are highly variable in amino acidsequence and probably in their tertiary structure. In addition tothis limitation, no direct structural information of the activatedstate of any GPCR is available. Consequently, an understandingof the conformational changes associated with receptor activa-tion has relied on biophysical and biochemical studies.

The development of the first published GnRH receptormolecular model was based on initial alignment and posi-tioning of the TM helices as indicated in the projection mapof the electron density of rhodopsin followed by refinementof the angles, kinking, and side chain orientation of the TMsbased on the specific amino acids comprising the GnRHreceptor TMs (18). The validity of the model and proposedinteractions of the TM side chains was tested by site-directedmutagenesis. An example is the observation that two resi-dues that are highly conserved in GPCRs, Asp2.50 in TM2 andAsn7.50 in TM7, appear to have undergone reciprocal muta-tion to Asn2.50(87) and Asp7.49(318) in the mouse GnRH receptor(Asp7.49(319) in human) (Figs. 7 and 8). This suggested that thetwo residues interact with each other. Mutation of Asn2.50(87)

in TM2 to Asp abolished receptor function, but a secondmutation in TM7, recreating the arrangement found in otherGPCRs [Asp2.50(87) and Asn7.49(318)], restored ligand binding(140). Cook et al. (141) reported that the reciprocal mutantwas totally inactive, but the original observation of goodbinding activity (140) has been confirmed (142–144). Thisrestoration of ligand binding by reciprocal mutation dem-onstrates that the side chains of two residues in TMs 2 and7 have complementary roles in maintaining the structure ofthe receptor and occupy the same microenvironment withinthe receptor helical bundle. This experimentally derived con-clusion was subsequently supported in the structural anal-


ysis of inactive rhodopsin, which shows that these residuesare capable of interacting through a water molecule (145).

Recently, the rhodopsin x-ray structure has been used asa template for homology modeling of the TM domains of theGnRH receptor (146) (Z.-L. Lu, unpublished observations).Evidence gleaned from the mutagenesis of the reciprocalTM2/TM7 mutations (mentioned above) and the disulfidebridges [Cys(14)/Cys5.27(200), Cys3.25(114)/Cys5.23(196), see be-low] was also used. A 2.5-nsec molecular dynamic simula-tion of the GnRH receptor in a water-vacuum-water box withno conformational restraints during the last 2 nsec was alsoundertaken. This revealed a hydrogen bond net of Glu2.53(90)-Lys3.32(121)-Asp2.61(98) between TM2 and TM3 that may rep-resent a component of intramolecular interactions that sta-bilize a receptor conformation similar to that of rhodopsin inthe inactive state (146).

The seven TM helical domains are known from physicalstructural studies in the rhodopsins to be arranged in a tightbundle enclosing a hydrophilic pocket and surrounded bythe hydrophobic membrane environment (18, 19, 25, 130,133–137) (see schematic in Fig. 10). The evolutionary con-servation of residues along a distinct face of the TM domainsin the various GnRH receptors is evident (compare Figs. 7and 8). This suggests that the conserved, more hydrophilicfaces are orientated toward the hydrophilic pocket or theboundaries formed by the seven TM domains and potentially

assists in the refinement of the molecular model. Thisproposal is supported by the studies on the TM2/TM7 in-teraction of Asn2.50(87) and Asp7.49(318) (140, 147) becauseAsn2.50(87) is clearly part of the conserved hydrophilic face ofTM2.

The relative positioning of TM3 and TM4 could be partlydeduced by the demonstration that Cys3.25(114) in EC1 andCys5.23(196) in EC2 form a disulfide bridge (Figs. 7, 8, and 10).This was determined by a combination of photoaffinity la-beling with a photoactive GnRH analog, followed by pro-tease digestion, reduction of S-S bonds and separation of thereceptor fragments by gel electrophoresis (148). The studyalso indicated that Cys(14) in the NH2-terminal domain andCys5.27(200) in EC2 form a second disulfide bridge, thus fur-ther defining the position of NH2 terminus and EC2 loopstructures. The highly conserved Trp4.50(164), located in themiddle of TM4, may make hydrogen or Van der Waals con-tacts with His2.45(82) (TM2) and Met3.42(131) (TM3) because theequivalent residues in rhodopsin form a H-bond network(130). Alanine mutation of these residues in the GnRH re-ceptor leads to a complete loss of ligand binding (Z.-L. Lu,unpublished observations), supporting the presence of thisintramolecular contact as a crucial interaction for positioningof TM2 and TM4, receptor folding, and stabilization of theground state. This TM2/TM4 interaction may be extended toTM3 via Van der Waals contact between His(82) (TM2) andMet(131) (TM3), sequestering TM3 within the bundle core andcreating a buttress against whose face the other TM helices,particularly TM6 and 7, can articulate and move (149). Theseintramolecular contacts may also account for TM3 having thegreatest tilt. The intracellular end of TM3 points into thecenter of the triangle formed by TMs 4, 5, and 6 of the receptorin the ground state (130). In addition, seven TM receptorssuch as rhodopsin may occur as dimers in native disc mem-branes (150). We find that TM4 of the GnRH receptor and anumber of other seven TM receptors contains theG/SxxxG/S motif, which is thought to favor TM helix-helixassociation (151, 152), suggesting that TM4 may form a sevenTM receptor homo- or heterodimer interface. Cysteine cross-linking between TM4s of the dopamine D2 receptors alsosuggested that the extracellular end of TM4 may form asymmetrical homodimer interface (153).

Although progress has been made in establishing molec-ular models of the TM helix bundle of the GnRH receptor, theproposed structure of the EC and IC is conjectural. Consid-erable effort has been directed at establishing programs todefine loop structures (e.g., based on sequences for loopstructures established from x-ray crystallography). How-ever, these are not applicable to large loop sequences. More-over, the known structures of the rhodopsin loops may bequite different from those of other GPCRs. Thus, a futurechallenge is the determination of the structure of the loops inthe GnRH receptors. Some progress has been made in cir-cular dichroism, NMR, and Raman spectral analysis of thestructure of a synthetic peptide of EC3 anchored by cross-links similar to the distance between the anchoring TM6 andTM7 domains (69). Contrary to the suggestion that EC3 hadan �-helical structure (154), these techniques revealed thepredominantly random structure of the loop. The NMR anal-ysis showed a low incidence of a �-hairpin structure (Fig. 11).

FIG. 10. A schematic representation of the human GnRH receptor.The receptor is viewed from above and shows the TM helices as acluster of cylinders (yellow, going into the page) that encompass thehydrophilic pocket and are surrounded by the light hydrophobic mem-brane environment. The TM helices are connected by the ECs (red).The dark bands represent the disulfide bridges stabilizing extracel-lular domains. The binding pocket is defined by some putative bindingsites, D2.61(98), N2.65(102), K3.32(121), N5.39(212), Y6.58(290), and D7.32(302) inthe receptor. [Adapted from R. P. Millar, Reproductive medicine: mo-lecular cellular and genetic fundamentals (edited by B. C. J. M.Fauser), Parthenon Publishing, Lancaster, UK, 2002, pp 199–224(21).]


When this structure is incorporated in the receptor model,Asp7.32(302) is able to interact with Arg8 of GnRH whendocked to the other interacting sites [Asp2.61(98), Asn2.65(102),and Lys3.32(121)] (Fig. 12). Due to the low sequence homologyof the EC and IC of the GnRH receptor with rhodopsin,Soderhall et al. (146) did not model the loops by using therhodopsin structure alone but also by homology modeling ofstructural motifs found in the Protein Databank. These mod-

els of loop structures provide a point of departure for ex-perimental testing. Despite the uncertainties of elements ofthe current GnRH molecular models, they are neverthe-less providing key insight into putative mechanisms of li-gand binding and receptor activation as a substrate forexperimentation.

Posttranslational modifications can also contribute to theoverall tertiary structure, stability, and expression of GPCRs.

FIG. 11. Interaction of Arg8 in GnRH with Asp7.32(302) of EC3 of the human GnRH receptor. The GnRH receptor model was based on therhodopsin structure and refined to accommodate known experimental data of interactions of TM domains. A �-hairpin conformation ofEC3 determined from the NMR structures of a cyclized EC3 peptide (69) was attached to TM6 and TM7 of the molecular model. Only TM6,EC3, and TM7 of the molecular model are shown for clarity. The GnRH molecule in its active �-II’ turned conformation has been dockedto Asp2.61(98), Asn2.65(102), and Lys3.32(121) cognate binding sites in the receptor, which are not shown for clarity. With these contacts inplace, Arg8 of GnRH is able to interact with Asp7.32(302) of the receptor as shown. [Adapted from R. Petry: J Med Chem 45:1026 –1034,2002 (69).]


FIG. 12. Molecular model of GnRH interactions with the human GnRH receptor (Z.-L. Lu, unpublished observations). GnRH was docked inthe �-II’ folded conformation (18, 54, 57) to the human GnRH receptor model built by homology modeling using the rhodopsin x-ray structureas a template. The model accommodates the experimentally determined or putative interactions of GnRH (black) and receptor (yellow/blue)residues [pGlu1 with Asn5.39(212); His2 with Asp2.61(98)/Lys3.32(121); Trp3 with Trp6.48(280); Tyr5 with Tyr6.58(290); Arg8 with Asp7.32(302); Pro9

with Trp2.64(101); and Gly10NH2 with Asn2.64(102)]. The hydrogen bonds are indicated by dashed lines. A, View of GnRH docked to its receptor.GnRH is shown in gray, the interacting residues of the receptor in yellow, and the seven TM helices in blue. B, Stereo view of the above model.The GnRH and receptor interacting residues are shown in white, and the seven TM helices in orange.


Glycosylation sites have been shown at Asn(4) and Asn(18) in themouse and Asn(18) in the human GnRH receptors (155, 156).Removal of these glycosylation sites decreases the number ofreceptors on the cell membrane presumably through impairedtrafficking of the receptor to the cell surface and/or stability(155, 156). Introduction of the additional mouse receptor gly-cosylation site in the human receptor increases receptor number(156). However, the removal or addition of glycosylation sitesdoes not affect receptor binding affinity or ligand selectivity(155, 156), indicating that glycosylation does not affect the over-all configuration of the receptor and the binding pocket.

IV. Binding of GnRH to the Mammalian Type IGnRH Receptor

Binding of ligand is a major component of receptor func-tion, and this interaction is the primary determinant ofwhether a receptor initiates signaling within the cell. Bindingof agonist ligands may be considered the first step in receptoractivation or receptor-mediated transduction of a hormonesignal across the cell membrane. Thus, understanding ligandbinding interactions is an important component of an un-derstanding of the fundamental mechanism of receptor func-tion. The binding of GnRH and GnRH agonist analogs tomammalian type I GnRH receptors has attracted consider-able experimental study and is reviewed in detail in thissection. However, the interactions of type I receptors withpeptide and nonpeptide antagonists and the ligand bindinginteractions of nonmammalian and type II GnRH receptorshave not been as well studied. Consequently, these are con-sidered in the next section in the context of what is knownabout GnRH binding to type I receptors.

Although site-directed mutagenesis has been a useful ex-perimental tool in defining ligand binding interactions ofGnRH receptors and most GPCRs, it is important to distin-guish direct receptor-ligand interactions from changes inreceptor structure or conformation that indirectly affect li-gand binding. One approach to this is to modify the ligandin parallel with receptor mutation (157). For example, if amutation is thought to decrease binding affinity by disrupt-ing a specific interaction with the ligand, then a ligand thatlacks the interacting group should have similar affinity forwild-type and mutant receptors. In the course of the targetedmutation of almost one third of all the amino acid residuesof the GnRH receptor, considerable advances have beenmade in identifying putative ligand contact sites in the mam-malian GnRH receptor (Figs. 7, 12, and 13A, and Table 3). Asis the case for other GPCRs that bind small peptides (158),amino acid residues in the ECs and exofacial parts of the TMhelices of GnRH receptors are thought to participate in ligandbinding interactions. Specific interactions of three residues,Asp2.61(98), Asn2.65(102), and Asp7.32(302), have been defined indetail, whereas residues Trp2.64(101), Lys3.32(121), Asn5.39(212),and Tyr6.58(290) have been shown to be important for bindingof agonist ligands but not antagonists, and specific interac-tions have been proposed for these residues. These residuesare discussed in numerical order, whereas other residues forwhich less experimental evidence is available are discussedat the end of this section.

A. Aspartate2.61(98) [D2.61(98)]

In mutating all extracellular acidic residues as putativeinteracting sites for Arg8 of GnRH, it was noted that mutationof Asp2.61(98) to Asn resulted in a large decrease in inositolphosphate production that was not consistent with an in-teraction with Arg8 (68). Interactions of Asp2.61(98) that con-tribute to high-affinity binding were investigated using acombination of site-directed mutagenesis of Asp2.61(98), li-gand modification, and computational modeling. The con-servative Asp2.61(98)Glu mutant exhibited marked decreasesin affinity for GnRH analogs containing the natural His2

amino acid and much smaller decreases for His2-substitutedGnRH analogs. Further analysis, using a series of analogswith different substitutions for His2, suggested that a hy-drogen bond is formed between Asp2.61(98) and the �-NHgroup of His2 (159). Substituting Asp2.61(98) with unchargedamino acids led to an additional decrease in affinity forGnRH (compared with the Asp2.61(98)Glu mutant) that didnot involve His2. It was concluded that the Asp2.61(98) sidechain has one or more charge-dependent interactions that areimportant for high-affinity binding, but distinct from theinteraction with His2. The computational model revealed anintramolecular salt bridge interaction of Asp2.61(98) in TM2with Lys3.32(121) in TM3, which positions Lys3.32(121) to forma hydrogen bond with the backbone C�0 group of Ser4 inGnRH (159). The model also identified a second potentialinteraction between Asp2.61(98) and the backbone NH groupof Trp3 (159). Interestingly, Lys3.32(121) had previously beenproposed to interact with His2 of GnRH (160) (see SectionVI.C). Thus, Asp2.61(98) appears to be involved in multipleinteractions with GnRH (His2, Trp3, and Ser4) as well as anintramolecular interaction with Lys3.32(121). These conclu-sions have been incorporated into a recent refined GnRHreceptor/ligand molecular model based on the crystal struc-ture of rhodopsin (Fig. 13A) (146).

B. Asparagine2.65(102) [N2.65(102)]

An investigation of the glycosylation of the GnRH receptorshowed that the Asn2.65(102) residue, located near the extra-cellular end of TM2, is not glycosylated, but enhanced po-tency of GnRH at the Asn2.65(102)Gln mutant suggested a rolein ligand binding (155). Mutation of Asn2.65(102) to Ala re-sulted in a 27- to 750-fold loss of potency in stimulatingphosphatidylinositol hydrolysis by GnRH and analogs con-taining the naturally occurring carboxyl-terminal Gly10-NH2(NH-CH2-CO-NH2). The mutation had a lesser effect on thepotency of analogs in which Gly10-NH2 was substituted withan ethylamide (-NH-CH2-CH3), and it was concluded thatAsn2.65(102) forms a hydrogen bond with Gly10-NH2 (161),probably via the C�O group (18). Although this conclusionis reasonable, the energy attributed to the loss of a hydrogenbond is insufficient to account for the 27- to 750-fold loss inpotency, and Asn2.65(102) may also be contributing to theconfiguration of the binding pocket. A subsequent studyconfirmed these findings and also showed that the bindingof an antagonist, which has d-Ala10-NH2 substituted forGly10-NH2, was decreased 2.8-fold by the Asn2.65(102)Ala mu-tation, consistent with possible disruption of a hydrogenbond with the C�O group of d-Ala10-NH2 (162).


C. Lysine3.32(121) [K3.32(121)]

The Asp residue that is highly conserved in TM3 of thebiogenic amine receptors interacts with the positivelycharged amine head group of biogenic amine ligands (157).We considered that the equivalent residue [Lys3.32(121)] of theGnRH receptor may interact with GnRH. Mutation ofLys3.32(121) to Arg had minor effects on ligand binding andagonist-stimulated inositol phosphate production, whereasmutation to Asp, Ala, or Leu led to a total loss of agonistbinding and inositol phosphate production. Mutation to Glnresulted in a 2000-fold reduction in agonist potency, withoutaffecting affinity for a peptide antagonist (160). BecauseGnRH has no negatively charged functional groups and be-cause peptide antagonists differ from agonists in their threeamino-terminal residues, Lys3.32(121) was proposed to interactwith His2 of GnRH by a charge-strengthened hydrogen bond(160). This proposal needs to be confirmed by systematicligand modification. The subsequent demonstration thatHis2 of GnRH interacts with Asp2.61(98), computational mod-eling that showed an interaction of Lys3.32(121) with Asp2.61(98)

and the similar phenotypes of mutants with uncharged sub-stitutions for Asp2.61(98) and Lys3.32(121), led to the suggestionthat Lys3.32(121) may have a role in maintaining the confor-mation of the agonist binding pocket and constraining thepeptide backbone of the receptor (159). If this is the case, thenit appears that GnRH peptide antagonist binding is not af-fected by these structural changes. Other computationalmodeling and mutagenesis studies suggested that Lys3.32(121)

also interacts with Glu2.53(90) of the receptor and pGlu1 ofGnRH (162). However, in more refined models, these authorssuggested that His2 interacts with both Asp2.61(98) andLys3.32(121) (146). At this stage, it is not clear whetherLys3.32(121) interacts with pGu1 or His2 or whether it has anydirect interaction with agonist ligands. The very large de-crease in agonist potency at the Lys3.32(121)Gln mutant (2000-fold) suggests disruption of the conformation of the ligandbinding pocket or multiple interactions.

D. Asparagine5.39(212) [N5.39(212)]

Mutation of Asn5.39(212) to Ala markedly reduced activityof both agonists and antagonists, but mutation to Gln de-creased agonist potency while having a minimal effect onantagonist interactions (162). This was interpreted as imply-ing that Asn5.39(212) forms part of the agonist binding pocket,and computational modeling showed an interaction of theAsn5.39(212) side chain with the backbone C�O group of His2

of an agonist and no interaction of Asn5.39(212) with an an-tagonist (162). The model was subsequently modified andshowed an interaction of Asn5.39(212) with pGlu1 (146, 163).

FIG. 13. Molecular models of a GnRH agonist and antagonist inter-actions with the human GnRH receptor (146). A, Starting from adistance of 30–40 Å outside of the defined binding pocket, the masscenters of interacting amino acid residues were restrained to grad-ually approach each other within a distance of 3–5 Å (146). After thesimulated annealing phase (5 psec heating up to 1500°K, 20 psec hotphase, and 25 psec slow cooling phase followed by complex minimi-zation), the lowest energy docked structure was selected. For D-Trp6

GnRH, the ligand was satisfactorily docked in the �-II’ turned con-formation to the identified contact sites in a putative active confor-mation of the receptor in which all of the experimentally identifiedbinding interactions are accommodated (146). D-Trp6 GnRH agonistinteractions with the activated receptor include a hydrogen bondbetween pGlu1 and Asn212; a hydrogen bond between His2 andLys3.32(121)/Asp2.61(98) that disrupts the hydrogen bond network ofGlu2.53(90)-Lys3.32(121)-Asp2.61(98); �-stacking between Trp3, Tyr5, andTrp6.48(280), Phe5.43(216); �-stacking between D-Trp6 and Trp6.57(289),which is close to the Cys14/Cys5.27(200) disulfide bridge; hydrogenbonds between Arg8 and Asp7.32(302) and Gly10NH2 and Asn2.61(102). A

�-II’ turn is formed by an intramolecular hydrogen bond between Tyr5

and Arg8 of the agonist. B, Cetorelix GnRH antagonist interactionswith the inactive receptor include a hydrogen bond between the NH2-terminal acetyl group and Asn5.39(212), �-stacking between D-Cpa andTrp6.48(280), a hydrogen bond between D-Pal3 and Lys3.32(121) withouteffect on the Glu2.53(90)/Lys3.32(121)/Asp2.61(98) hydrogen bond network,D-Cit6 near the Cys14/Cys5.27(200) disulfide bridge, hydrogen bondsbetween Arg8 and Asp7.32(302) and D-Ala10NH2 and Asn2.65(102). A �-II’turn is formed between Tyr5 and Arg8 of the antagonist. [Adaptedwith permission from Ref. 146.]


TABLE 3. Summary of effects of point mutations on GnRH receptor function

Mutation Expression GnRH affinity Coupling efficiency/Emax Function, comments Ref no.

H Glu8Gln No effect 68H Asn10Lys Decreased Decreased No effect Rescued by IN-3 221, 283H Cys14Ser Undetectable Decreased 148R Cys14Ala Decreased Decreased 284H Thr32Ile Decreased Undetectable Rescued by IN-3 218, 283H Lys36Ala No effect 162H Asn1.50(53)Aspa Decreased Undetectable Decreased Expression, activation 143H Asn1.50(53)Ala Decreased Undetectable Decreased Expression, activation 143H Asn1.50(53)Leu Decreased Undetectable Decreased Expression, activation 143M Leu1.55(58)Ala Decreased No effect Decreased cAMP cAMP coupling 239M Lys1.56(59)Gln Decreased No effect No effect 239M Gln1.58(61)Glu Decreased No effect No effect 239M Lys1.59(62)Gln Decreased No effect No effect 239M Leu2.36(73)Arg Increased No effect Decreased cAMP cAMP coupling 239M Ser2.37(74)Glu Decreased No effect Decreased Coupling 239M Leu2.43(80)Ala Decreased No effect Decreased Coupling 239M Asn2.50(87)Asp Undetectable Undetectable Expression, activation 140, 144H Asn2.50(87)Ala Decreased Undetectable Undetectable Expression, activation 143, 162H Asn2.50(87)Asp Decreased Undetectable Undetectable Expression, activation 143H Asn2.50(87)Gln Decreased Undetectable Very low Expression, activation 143Cf Asp2.50(90)Asn Decreased Undetectable Undetectable 168H Glu2.53(90)Lys Decreased Rescued by IN-3 220, 283, 285H Glu2.53(90)Ala Undetectable Undetectable 162M Glu2.53(90)Gln No effect 68H Asp2.61(98)Ala Undetectable Undetectable Binding, His2 162H Asp2.61(98)Ala Decreased Decreased Decreased Binding, His2 159H Asp2.61(98)Val Decreased Decreased Decreased Binding, His2 159H Asp2.61(98)Asn Decreased Decreased Decreased Binding, His2 159H Asp2.61(98)Glu Decreased Decreased Decreased Binding, His2 159H Trp2.64(101)Ala Decreased Decreased Binding, agonist 162H Asn2.65(102)Ala No effect Decreased Binding, Gly10-NH2 161, 162H Asn2.65(102)Gln Increased Binding, Gly10-NH2 155H Gln2.69(106)Arg Decreased Decreased Rescued by IN-3 167, 213, 283M Glu2.74(111)Gln No effect 68R Cys3.25(114)Ala Undetectable Undetectable 284H Lys3.32(121)Arg Decreased No effect No effect Binding, agonist 160H Lys3.32(121)Gln Undetectable Undetectable Decreased Binding, agonist 160H Lys3.32(121)Leu Undetectable Undetectable Undetectable Binding, agonist 160M Lys3.32(121)Asp Undetectable Undetectable Undetectable Binding, agonist 160Cf Lys3.32(124)Met Decreased Decreased Decreased Binding, expression 168H Ala3.40(129)Asp Decreased Undetectable Undetectable Rescued by IN-3 167, 215, 283H Ile3.46(135)Ala Undetectable Undetectable Undetectable 193H Ile3.46(135)Val Undetectable Undetectable Undetectable 193H Ile3.46(135)Leu Decreased No effect Increased 193M Asp3.49(138)Ala Undetectable Undetectable Undetectable 193M Asp3.49(138)Glu Decreased No effect Increased 194M Asp3.49(138)Asn Decreased No effect Increased 193, 194M Arg3.50(139)Lys Undetectable Undetectable Undetectable 193M Arg3.50(139)Gln Increased Decreased Decreased 193, 194M Arg3.50(139)Ala Increased Decreased Decreased 194M Arg3.50(139)Ser Increased Decreased Decreased 194M Arg3.50(139)His Undetectable Undetectable Undetectable 193H Arg3.50(139)His Undetectable Undetectable Rescued by IN-3 221, 283M Ser3.51(140)Tyr No effect Increased No effect 247M Ser3.51(140)Ala No effect No effect No effect 194H Ile3.54(143)Ala No effect Decreased Decreased 193H Ile3.54(143)Leu Decreased No effect Increased 193H Ile3.54(143)Val Decreased No effect No effect 193M Leu3.58(147)Ala No effect No effect Decreased 247M Leu3.58(147)Asp No effect No effect Decreased 247H Ser4.54(168)Arg No effect Undetectable Undetectable Not rescued by IN-3 167, 219H Ala4.57(171)Thr Undetectable Undetectable 286H Arg4.65(179)Ala Undetectable Undetectable Undetectable 162M Asp4.71(185)Asn No effect 68H Lys4.77(191)Arg No effect No effect 287H Lys4.77(191)Glu No effect No effect 287H Lys4.77(191)Gln No effect No effect 287H Lys4.77(191)Ala No effect No effect 287H Lys4.77(191) del Increased Increased Increased 287H Cys5.23(196)Ser Undetectable Undetectable 148


TABLE 3. Continued


R Cys5.23(195)Ala Undetectable Undetectable 284F Ala5.25(201)Thr Increased 115H Cys5.27(200)Ser Undetectable Decreased 148R Cys5.27(199)Ala Decreased Decreased 284H Cys5.27(200)Tyr Decreased Undetectable Rescued by IN-3 218, 283, 285H Gln5.31(204)Ala No effect No effect 162H Trp5.32(205)Ala Decreased No effect 162H Trp5.33(206)Ala Undetectable Undetectable Undetectable 162H His5.34(207)Ala Decreased No effect 162H Gln5.35(208)Ala No effect No effect 162F Lys5.35(211)Glu Decreased 115H Phe5.37(210)Ala Increased No effect 162H Tyr5.38(211)Ala Undetectable Undetectable Undetectable 162H Asn5.39(212)Ala Decreased Decreased Binding, agonists and

antagonists162

H Asn5.39(212)Gln Decreased Decreased Binding, agonists 162H Phe5.40(213)Ala Decreased Decreased NR 162H Phe5.41(214)Ala Undetectable Undetectable Undetectable 162H Thr5.42(215)Ala Undetectable Undetectable Undetectable 162H Phe5.43(216)Ala Decreased No effect 162H Ser5.44(217)Ala Decreased No effect 162H Ser5.44(217)Arg Decreased Undetectable Not rescued by IN-3 216, 283H Met5.54(227)Thr No effect No effect 175M Leu5.65(237)Ile No effect No effect Decreased Coupling, expression 288M Leu5.65(237)Val Decreased No effect Decreased 288M Leu5.65(237)Ala Undetectable Undetectable Undetectable 288M Leu5.65(237)Arg Undetectable Undetectable Undetectable 288M Leu5.65(237)Asp Undetectable Undetectable Undetectable 288R Thr5.66(238)Ala Decreased Decreased Decreased 289R Ser5.81(253)Ala Decreased No effect No effect 289H Ala6.29(261)Leu Decreased Coupling 243H Ala6.29(261)Ile Decreased Coupling 243H Ala6.29(261)Lys No effect Decreased Coupling 243H Ala6.29(261)Glu Decreased Coupling 243H Ala6.29(261)Phe Decreased Coupling 243H Ala6.29(261)Gly No effect Unchanged Coupling 243H Ala6.29(261)Pro Decreased Coupling 243H Ala6.29(261)Ser No effect No effect 243H Ala6.29(261)Val No effect Decreased Coupling 243H Arg6.30(262)Gln No effect No effect Decreased Coupling, rescued by

IN-3167, 213, 283

F Thr6.31(269)Met Increased 115R Thr6.33(2.64)Ala Decreased No effect No effect 289H Leu6.34(266)Arg Decreased Undetectable Rescued by IN-3 167, 218,

283, 285,290

H Phe6.40(272)Leu Increased No effect No effect 291H Phe6.40(272)Tyr Decreased No effect No effect 291H Phe6.40(272)Glu Undetectable Undetectable 291H Phe6.40(272)Lys Undetectable Undetectable 291H Phe6.44(276)Leu Undetectable Undetectable 291H Phe6.44(276)Tyr Decreased No effect No effect 291H Cys6.47(279)Tyr Decreased Undetectable Rescued by IN-3 283, 285R Trp6.48(279)Ser Decreased Decreased Undetectable 164R Trp6.48(279)Arg Undetectable Undetectable Undetectable Rescued by

Val6.68(299)Ala164

H Tyr6.51(283)Ala Undetectable Undetectable 163H Tyr6.52(284)Ala Undetectable Undetectable 163H Tyr6.52(284)Cys Decreased Decreased Rescued by IN-3 283, 292F Leu6.52(290)Phe Increased 115H Trp6.57(289)Ala Decreased Decreased No effect 163H Tyr6.58(290)Ala Decreased Decreased No effect 163H Trp6.59(291)Ala Undetectable Undetectable 163H Phe6.60(292)Ala No effect No effect No effect 163M Asp6.61(292)Asn No effect 68F Phe6.61(299)Tyr Decreased 115M Glu6.63(294)Gln No effect 68R Val6.68(299)Ala Undetectable Undetectable Undetectable Rescued by

Trp6.48(279)Arg164

M Glu7.32(301)Gln Decreased Decreased Binding, Arg8 68H Asp7.32(302)Asn Decreased Decreased Binding, Arg8 70

Continued on next page


The latter proposal is similar to a previously reported modelof GnRH binding to the rat GnRH receptor, in which pGlu1

lies at the central cleft in the neighborhood of this Asn5.39(212)

in TM5 (164). Further studies with systematically substitutedGnRH analogs and Asn5.39(212) mutants are required to definethe role of Asn5.39(212) in ligand recognition. It should benoted that the decreased activity of both agonists and an-tagonists at the Asn5.39(212)Ala mutant suggests an additionalrole for the Asn212 side chain in binding antagonists or inreceptor structure.

E. Tyrosine6.58(290) [Y6.58(290)]

Mutation of aromatic amino acids at the extracellular endof TM6 [Tyr6.51(283), Tyr6.52(284), Trp6.57(289), Tyr6.58(290),Trp6.59(291), Phe6.60(292)] to Ala revealed that the Tyr6.51(283),Tyr6.52(284), and Trp6.59(291) mutants were totally inactive andthe Phe6.60(292) mutant was fully active. The Trp6.57(289) andTyr6.58(290) mutants had reduced expression, and both mu-tants retained wild-type antagonist binding affinity. TheTrp6.57(289) mutant showed a decrease in agonist potency(�10-fold) in a signaling assay, whereas the Tyr6.58(290) mu-tant showed markedly decreased potency (200- to 1000-fold)of agonists (163). This effect of mutation of Tyr6.58(290) to Alahas been independently verified (J. S. Davidson, J. Hapgood,and R. P. Millar, unpublished observations). On the basis ofdocking the ligand to the receptor model, it was proposedthat Tyr5 of GnRH interacts with Tyr6.58(290) (163). Experi-mental evidence with Tyr5-substituted GnRH analogs is re-quired to test this proposal.

F. Aspartate7.32(302) [D7.32(302)]

As described above, mammalian type I GnRH receptors pref-erentially bind mammalian GnRH, which has a positively

charged Arg residue in position 8 (18, 52). Substituting Arg8

markedly decreased peptide affinity for these receptors (52). Toinvestigate the possibility that Arg8 forms an electrostatic in-teraction with an acidic residue in the receptor, conserved acidicresidues of the mouse GnRH receptor were mutated to theisosteric amide residues, Asn or Gln, and a mutant that did notdiscriminate Arg8-containing GnRH from uncharged[Gln8]GnRH was sought. One mutant, Glu7.32(301)Gln, had de-creased affinity for mammalian GnRH, but did not changeaffinity for [Gln8]GnRH and increased affinity for the nega-tively charged [Glu8]GnRH, showing a loss of selectivity forArg8 and gain of function for [Glu8]GnRH (68). Similarly, mu-tation of the equivalent residue of the human GnRH receptor[Asp7.32(302)] to Asn showed that the acidic Asp7.32(302) residueconfers selectivity of the human receptor for Arg8 (70). How-ever, although Arg8 and the acidic residue in EC3 are requiredfor high-affinity binding of GnRH, conformationally con-strained GnRH analogs (see Section II) bound the receptor withhigh affinity that was independent of Arg8 and/or the acidicresidue (68, 70). This result indicates that once the ligand is inthe high-affinity conformation, the putative interaction of Arg8

with the acidic residue does not contribute to the binding en-ergy of the final ligand-receptor complex and suggests that theacidic residue induces or selects a �-II’ conformation of theligand. This observation led to the proposal that Arg8 of GnRHinteracts transiently with the acidic residue to induce a high-affinity conformation of the ligand that allows it to interact witha final binding pocket, which does not include the acidic residue(70). These results suggest that caution should be exercised inusing the Arg8 interaction with Asp7.32(302) as a fixed point incomputational models of ligand-receptor complexes.

Although nonmammalian type I GnRH receptors do notpreferentially bind Arg8-containing mammalian GnRH, theacidic residue in EC3 is conserved in these receptors (Fig. 8).

TABLE 3. Continued


Cf Asp7.32(304)Glu No effect No effect No effect Binding, Arg8 53Cf Asp7.32(304)Asn Decreased Decreased No effect Binding, Arg8 53Cf Asp7.32(304)Ala No effect Decreased No effect Binding, Arg8 53H Pro7.33(303)Ala No effect Decreased Decreased Binding, Arg8, indirect 165, 165aH Phe7.43(313)Leu No effect No effect Binding, nonpeptide

agonist175

D Leu7.43(313)Phe No effect No effect Binding, nonpeptideantagonist

175

M Asp7.49(318)Asn Decreased Decreased 140H Asp7.49(319)Asn Decreased No effect Decreased 143H Asp7.49(319)Glu Decreased No effect Decreased 143H Asp7.49(319)Ala No effect No effect Decreased 143H Asp7.49(319)Leu Decreased Undetectable Low 143M Tyr7.53(322)Ala Decreased Coupling 144M Tyr7.53(322)Phe No effect 144R Phe7.56(325)Ala Undetectable Undetectable 256R Ser7.57(326)Ala No effect Decreased Decreased 256R Ser7.57(326)Thr No effect Decreased No effect 256R Ser7.57(326)Val No effect Decreased No effect 256R Ser7.57(326)Tyr No effect Decreased No effect 256

Cf, Catfish; D, dog; F, frog; H, human; IN-3, nonpeptide antagonist; M, mouse; R, rat.a The consensus numbering system of Ballesteros and Weinstein (293) is used to allow easy comparison of equivalent residues in GPCRs

of different lengths. The most conserved residues in each TM segment (Fig. 7) are assigned the number 50, and other residues are numberedrelative to these residues. For example, the Lys residue in TM3 that corresponds to the Asp residue, which is important for ligand binding inmonamine receptors, is designated Lys3.32(121) in mammalian GnRH receptors and Lys(3.32)124 in the catfish receptor, because it is located 18residues amino-terminal of the most conserved residue in TM3, Arg3.50(139).


In mammalian receptors, the acidic residue is followed by aPro residue, whereas Pro precedes the acidic residue in non-mammalian receptors. The unique structural characteristicsof Pro residues suggested that the position of Pro7.33(303) maybe important for the selective binding of mammalian GnRHby mammalian receptors. Substituting Pro7.33(303) of the hu-man GnRH receptor or introducing Pro into position 301,preceding Asp7.32(302), decreased receptor affinity for GnRH,but not for analogs lacking Arg8 (165). Secondary structureprediction showed that modifying Pro7.33(303) influences theconformation of EC3 (165, 165a). These results show thatPro7.33(303) may stabilize a conformation of EC3 of mamma-lian GnRH receptors that orients the acidic side chain toallow selective binding of mammalian GnRH.

G. Effects of mutations of other residues on the ligandbinding pocket

Other receptor residues, for which less experimentalevidence is available, have been proposed to be involvedin ligand binding. Mutation of Trp2.64(101) to Ala resulted ina pronounced shift in agonist-induced signal transduction(162). This residue is adjacent to Asn2.65(102), which is thoughtto interact with Gly10NH2 of GnRH (161). The molecularmodel suggests that Trp2.64(101) makes a hydrogen bond withthe oxygen of the Leu7 backbone of a GnRH agonist (162).However, the loss of energy associated with loss of a singlehydrogen bond in the mutant cannot account for the shift inagonist-induced signal transduction of three orders of mag-nitude, so it is likely that mutation of Trp2.64(101) has a majoreffect on the formation of the binding pocket for agonists andnot antagonists. Phe5.43(216) mutation to Ala had little effecton receptor function, but mutation to Tyr decreased activityabout 10-fold (162). It is likely that Phe5.43(216) does not playan important role in ligand binding, but insertion of a phe-nolic residue disrupts receptor function.

Three computational models have proposed thatTrp6.48(280) [Trp6.48(279) in the rat] in TM6 interacts with Trp (3)of GnRH (146, 164, 166). The Trp6.48 residue is also proposedto interact with a Val residue in EC3 (164) or a conserved Pheresidue in TM7 (166). Mutation of Trp6.48 to Ser or Arg de-creased ligand binding and abolished inositol phosphateproduction. The Trp6.48 residue is highly conserved amongrhodopsin-like GPCRs, and the loss in signal transduction isconsistent with a conserved role in receptor activation ratherthan specific ligand interactions.

Many mutations have been found to have no affect onligand binding (Table 3). These residues are most probablyfiller residues that play little or no role in receptor function.In contrast, many other mutations (Table 3) resulted in com-plete loss of receptor function. These findings are not in-structive because the loss of function, which may be due todestruction of the overall receptor architecture and/or bind-ing pocket, failure to target to the plasma membrane, orinstability of the receptor at the cell surface and rapid tar-geting to the proteosome, makes it difficult to measure pa-rameters of ligand binding. The use of small molecule an-tagonists to rescue these mutants (167) may assist indetermining their binding and signaling characteristics.

H. Ligand docking to the receptor

GnRH and GnRH agonists have been satisfactorily dockedto receptor models via the identified binding sites (18, 159,162–164). In the most recent GnRH receptor model, d-Trp6

GnRH was docked in the �-II’ turned conformation to theidentified contact sites in a putative active conformation ofthe receptor in which all of the experimentally identifiedbinding interactions are accommodated (Fig. 13A) (146). Inthis model the Glu2.53(90)-Lys3.32(121)-Asp2.61(90) hydrogenbond net, identified in a previous model (163), is disturbedby His2 hydrogen bonds with Lys3.32(121) and Asp2.61(98),which are proposed to be involved in receptor activation (seeSection VI). In addition to the interacting sites for the naturalGnRH agonist, d-Trp6 of the superactive agonist was pro-posed to interact with Trp6.57(289), adjacent to Tyr6.58(290),which is proposed to interact with Tyr5 (146). The proposedinteraction of the d-Trp6 side chain with Trp6.57(289) is notconsistent with experimental results in which mutation ofTrp6.57(289) to Ala led to a smaller decrease in potency of[d-Trp6]GnRH (5.9-fold) than was found for GnRH (16-fold)and d-Ala6-substituted analogs (8-fold) (163). During thesimulation, the C� atoms of the receptor were restrictedusing harmonic restraints of 1 kcal mol�1 Å�2 that allow onlysmall conformational changes of the receptor model (163),and therefore the model, in general, represents the dockingof GnRH analogs to the inactive state of the receptor. How-ever, agonist peptide binding to its receptor would lead toconformational changes of both ligand and receptor.

V. Binding Interactions of Other GnRH Ligands andOther Receptors

A. GnRH II

The presence of GnRH II in man (48), together with anapparent absence of a functional full-length type II receptor(45, 86) and high binding affinity of GnRH II for the type IGnRH receptor (70–72), suggests that this receptor hasadopted the role of the cognate receptor for GnRH II (45). Asfor GnRH I, mutation of Asp2.61(98) (159) and Asn2.65(102) (161)decreased potency of GnRH II, and these residues appear tobe contact sites for GnRH II. Although GnRH II activity wasnot measured at the Lys3.32(121)Gln mutant, decreased po-tency of GnRH II with mutation of the equivalent Lys[Lys3.32(124)] in the catfish receptor suggests that Lys3.32(121) isimportant for GnRH II binding (168). However, Asp7.32(302),which confers specificity for Arg8 of GnRH I, is not requiredfor high-affinity binding of GnRH II (68, 70, 72). This is notunexpected because Asp7.32(302) interaction with Arg8 ofGnRH I is thought to represent an initial tethering requiredto configure GnRH I in the folded conformation for inter-action with the other binding sites (68, 70). In GnRH I analogsconstrained in the folded conformation with d-amino acidsubstitution for Gly6, the interaction of Arg8 and Asp7.32(302)

is not required for high-affinity binding (68, 70). Evidence hasbeen presented that GnRH II is already stabilized in thefolded conformation because neutral d-amino acid substitu-tion does not enhance binding affinity (R. P. Millar, unpub-lished observations). Thus the lack of requirement of aninteraction of GnRH II with Asp7.32(302) is expected.


B. Peptide agonists

As mentioned above, two of the most common modifica-tions incorporated into synthetic peptide agonists are sub-stitution of Gly6 with a d-amino acid and Gly10.NH2 withethylamide. Both of these modifications affect receptor in-teractions that have been defined for GnRH. Mutagenesisand ligand modification studies have indicated that GnRHagonists with d-amino acid substitutions for Gly6 and withno substitution of Gly10.NH2 are dependent on Asp2.61(98),Asn2.65(102), and Lys3.32(121) for binding and receptor activa-tion (159–161, 163). Because Asp7.32(302) plays a role in inter-acting with Arg8 for configuring the native ligand at thereceptor, this residue is not required in agonists that areconfigured by the d-amino acid substitution of Gly6 (68, 70).These experimentally derived conclusions contrast with mo-lecular models in which Arg8 is modeled to interact withAsp7.32(302) for the d-Trp6 GnRH agonist (146, 162) (Fig. 13A).GnRH agonists with ethylamide substitutions for Gly10.NH2show smaller decreases in potency at the Asn2.65(102)Ala mu-tant (161, 162) and probably do not interact with Asn2.65(102),whereas GnRH agonists that retain Gly10.NH2, retain normalinteraction with Asn2.65(102) (Fig. 13A).

C. Peptide antagonists

Identification of binding sites for GnRH peptide antag-onists has been less comprehensive than for agonists. LikeGnRH, peptide antagonists of the GnRH receptor are de-capeptides, but with 50 –70% of amino acids substituted(Fig. 5), and they exhibit classical competitive antagonism.This would suggest that antagonists occupy binding sitesthat differ from but overlap the agonist binding pocket.Recent proposals that agonist and antagonist or inverseagonist ligands bind distinct active and inactive confor-mations of the GPCRs (169) suggest that competitive an-tagonism may occur without any overlap of agonist andantagonist binding sites.

Evidence for differences between the ligand bindingsites of agonists and antagonists was provided by earlybiochemical studies. Pretreatment of pituitary membraneswith proteolytic enzymes decreased binding of labeledantagonist more than that of labeled agonist. This indi-cated that the agonist binding site is less accessible andmore buried within the receptor molecule than the antag-onist binding site (170). Differences in the binding sites arealso suggested by the greater effects of monovalent anddivalent cations on agonist binding (170, 171). In anotherstudy, tryptic digestion of GnRH receptors that had beenphotoaffinity labeled with agonist or antagonist yieldeddifferent size fragments, suggesting distinct sites of at-tachment for agonist and antagonist ligands (172) or dis-tinct configurations of the agonist- and antagonist-boundreceptor. In this study, the photoactive agent was attachedto d-Lys in position 6 of the ligands. In contrast, identicallabeled receptor fragments were obtained for labeling withboth a photoactive agonist (148) and a photoactive antag-onist (173) when the receptor was digested with GluC totarget specific cleavage sites at Glu residues. Despite thephotoactive group being at position 1 of the antagonist and

position 6 of the agonist, both peptides were cross-linkedto Cys14 in the receptor. The identification of the samereceptor residue in cross-linking two peptides with pho-toactive groups at different positions is consistent with thepeptides having distinct binding configurations. This find-ing is also consistent with the concept that agonists, in-verse agonists, and antagonists bind to different receptorconformations representing the continuum of equilibriabetween states of the active and inactive receptor. How-ever, it may also suggest that there is considerable move-ment of the ligand and that covalent attachment is to themost photoreactive amino acid in the general environment(173). Thus, this kind of photoaffinity labeling may notidentify precise ligand interaction sites.

The first mutation displaying differential effects on agonistand antagonist interactions was the marked effect of muta-tion of Lys3.32(121) to Gln on agonist potency and its failure toaffect antagonist affinity. This indicated that, althoughLys3.32(121) may be important for agonist binding, it clearly isnot an antagonist contact site (160). Other mutations thathave been shown to have differential effects on agonistand antagonist interactions include Asp2.61(98)Glu,Trp2.64(101)Ala, Asn5.39(212)Ala, Asn5.39(212)Gln, Tyr6.58(290)Ala,and Asp7.32(302)Asn (70, 159, 162, 163). The Asp2.61(98)Glu mu-tation disrupted interaction with His2 of GnRH, a residuethat is substituted with bulky d-amino acids in antagonists,so its minimal disruption of Cetrorelix binding (159) was tobe expected. The Asp2.61(98) side chain is unlikely to interactwith antagonists. The Trp2.64(101)Ala mutation decreased ag-onist potency by three orders of magnitude, but also de-creased antagonist binding affinity 23-fold (162). This effecton antagonist binding may result from disruption of a directinteraction of Trp2.64(101) with the antagonist, but disruptionof receptor configuration is more likely. Mutating Asn5.39(212)

to Gln had minimal effects on antagonist interactions, but theAla mutation decreased antagonist affinity 86-fold (162). Thislarge change suggests that hydrophilic interactions at theAsn5.39(212) locus stabilize the configuration of the antagonistbinding site. The Asp7.32(302)Asn mutation showed little or noeffect on binding of antagonists, including two that have Argin position 8 (70) (K. D. Pfleger, and R. P. Millar, unpublishedobservations). This is consistent with the proposed role ofAsp7.32(302) in inducing a high-affinity ligand conformation(70). Because peptide antagonists are constrained in the high-affinity conformation, they are unlikely to interact directlywith Asp7.32(302).

Docking of peptide antagonists to a GnRH receptormolecular model suggested that antagonists with Arg inposition 8 [Cetrorelix (Fig. 5)] and a dicyclic peptide (174)interact with Asp7.32(302) as for Arg8 agonists (146). Theseresearchers also postulated an interaction of Asn2.65(102)

with the C-terminal amide as in native GnRH (Fig. 13B).However, these interactions were not demonstrated ex-perimentally. These researchers also propose that the NH2terminal CO moiety of Cetrorelix interacts withAsn5.39(212), d-Pal3 with Lys3.32(121), d-Cpa2 with Trp6.48(280)

and d-Cit6 with the Cys (14) Cys5.27(200) disulfide bridge(Fig. 13B). The dicyclic peptide NH2-terminal CO isthought to also interact with Asn5.39(212), whereas d-Cpa3

interacts with Trp6.48(280). Although the catfish GnRH re-


ceptor has low affinity for GnRH antagonist 135–18 (seeSection IV.D), substitution of EC 1, 2, and 3 of the catfishGnRH receptor into the human receptor had little effect onthe affinity of antagonist binding (K. D. Pfleger, and R. P.Millar, unpublished observations). This suggests that thisGnRH antagonist either binds to TM residues or interactswith the few EC loop residues that are conserved betweencatfish and human receptors (Fig. 8).

D. Nonpeptide antagonists

The endeavor to develop orally active nonpeptide antag-onists for the treatment of hormone-dependent diseases andfor new generation contraceptives (14) has resulted in thedevelopment of molecules with nanomolar binding affinities(Fig. 6). Although generally less comprehensive studies havebeen conducted on identifying their binding sites, some in-dications have arisen from ligand docking to molecular mod-els and limited mutagenesis studies.

A quinolone-based antagonist (compound 7, Fig. 6) boundthe dog GnRH receptor with a 160-fold decreased affinitycompared with the human receptor (175). Construction ofdog/human chimeric receptors followed by site-directedmutagenesis revealed that Phe7.43(313) in TM7 of the humanreceptor (Leu in the dog) is responsible for the difference inaffinity. Peptide agonist and antagonist analog binding wasunaffected by mutation of this residue. Docking the quino-lone antagonist to the human and dog receptor modelsshowed that the quinolone ring faces Phe7.43(313) andLeu7.43(313), respectively, and the difference in surface area ofthese two side chains is 90 Å, which contributes 2.25 kcal/mol and accounts for the difference in binding affinity. Thebinding model also predicted that GnRH binding sitesLys3.32(121) and Asp7.32(302) interact with the compound andthat its binding site overlaps that of GnRH. However, nodirect experimental evidence (e.g., with mutant receptors)has been presented for the interaction of the quinolone an-tagonist with these GnRH binding sites.

A thienopyridine-based antagonist (compound 5, Fig. 6)with nanomolar affinity for the human receptor was alsodocked to a human GnRH receptor model (92). This pro-poses a hydrophobic lining to the bottom part of the bind-ing pocket that interacts with difluorobenzyl and thethienopyridine moieties and an interaction of the posi-tively charged amino groups with Asp7.32(302) (Fig. 6).Again, no mutagenesis studies were reported to supportthe proposed interactions. However, this laboratory dem-onstrated that the mutant Asp7.32(302)Asn exhibited a 5-foldreduction in binding affinity for the compound (R. P.Millar, unpublished observations).

Overall, the current knowledge on binding of nonpeptidesmall molecule GnRH antagonists indicates that the bindingpocket partially overlaps that of GnRH and may also useresidues crucial for peptide binding. Unlike the GnRH pep-tide, which predictably interacts with the same residues inmammalian GnRH receptors, some of the nonpeptide an-tagonists show marked species specificity (binding affinitydifferences of several orders of magnitude) despite sequenceidentity exceeding 80%. Thus, small microdomain differ-ences such as Phe313 in the human receptor can result in large

species differences in binding affinities of nonpeptide an-tagonists, as has been noted in a number of neuropeptideGPCRs.

E. Binding sites in nonmammalian type I GnRH receptors

The conservation of all the well-established and putativeligand binding sites [Asp2.61(98), Trp2.64(101), Asn2.65(102),Lys3.32(121), Asn5.39(212), Tyr6.58(290), and Asp7.32(302)] of the hu-man type I receptor suggests that these residues serve thesame function (see equivalent residues in Fig. 8). However,their functional significance has only been partially investi-gated and only in the chicken and catfish receptors (53)(K. D. Pfleger, unpublished observations). Although anacidic residue is present in the equivalent position of thehuman Asp7.32(302) in the nonmammalian type I receptors,this acidic residue would not be expected to interact withArg8 of mammalian GnRH because these receptors do notbind mammalian GnRH with higher affinity than the othervertebrate GnRHs as occurs for the mammalian type I re-ceptor (51, 66, 67, 71, 119, 176, 177). It was observed that a Proresidue precedes the acidic residue in EC3 of the nonmam-malian type I GnRH receptors, whereas the Pro residue fol-lows the acidic residue in mammalian type I receptors. Thissuggests that the orientation of the side chain of the acidicresidue may effect selectivity for Arg8 GnRH. The recentdemonstration that exchange of the Pro residue to the non-mammalian position in the human receptor resulted in a lossof selectivity for mammalian GnRH in the human receptorsuggests that a Pro residue following the acidic residue isnecessary for selective binding of mammalian GnRH bymammalian receptors (165). It has been reported that mu-tation of the acidic residue of the catfish GnRH receptordecreased affinity for mammalian GnRH and a series ofArg8-containing analogs, including one with a d-amino acidin position 6 (53). Because the catfish receptor has very lowaffinity for mammalian GnRH, this result suggests that evenif Arg8 and the acidic residue do interact, the catfish receptor,unlike the mammalian receptor, is unable to induce a high-affinity conformation of mammalian GnRH. The ligand bind-ing sites of the nonmammalian GnRH receptor may be con-figured differently from the nonmammalian receptor ligandbinding sites. This is reflected in the 100-fold higher bindingaffinity of the catfish, chicken, and Xenopus receptors forGnRH II compared with the human GnRH receptor, despitethe apparent use of the same binding sites.

F. Binding sites in type II receptors

With the exception of Asp7.32(302), all of the proposedGnRH binding sites for type I receptors are present in typeII receptors (Fig. 8). These conserved sites are likely, there-fore, to be involved in binding of the cognate ligand, GnRHII. The absence of Asp7.32(302) is expected because GnRH IIlacks Arg8 and mutation of the acidic residue in the mouseand human type I receptors did not affect the binding ofGnRH II (68, 70). The reduced binding affinity of mammalianGnRH at the marmoset (83), bullfrog (67), and Xenopus (B.Troskie, unpublished observations) type II receptors is prob-ably partly due to the absence of Asp7.32(302), which is im-


portant for interaction with Arg8 of GnRH and high-affinitybinding. Recent mutations of the marmoset type II receptorresidues equivalent to Asp2.61(98), Asn2.64(102), and Lys3.32(121)

show that the functions of these amino acids are similar intype I and II GnRH receptors (C. A. Flanagan, unpublishedobservations). The high-affinity binding of GnRH II to typeII receptors and all other GnRH receptors may be due to itsstabilization in the �-II’ turn conformation due to intramo-lecular interactions (see Section V.A.).

G. Utilization of binding sites common to the rhodopsinfamily of GPCRs

There appears to be significant conservation of the bindingsites for GnRH and those of other rhodopsin family GPCRs,albeit with alterations in the precise positioning and natureof the interacting residues (Table 4). Asp2.61(98) of the GnRHreceptor is equivalent to functionally important residuesGln2.61(108) in the closely related vasopressin V1a receptor, andVal2.61(81) and Phe2.61(91) of the D2- and D4-dopamine recep-tors. Trp2.64(101) of the GnRH receptor has critical equivalentsPhe2.64(86), His2.64(93), and Thr2.64(134) in the �1-adrenergic, �2-adrenergic, and 5-hydroxytryptamine (2A) (5HT2A) recep-tors, respectively. Lys3.32(121) in the GnRH receptor is foundin the homologous position to the Asp3.32 residue, which actsas the counter-ion of the amine group of small biogenicamines in the adrenergic-, muscarinic-, acetylcholine-, his-tamine-, dopamine-, and 5-hydroxytryptamine (2A) (5HT2A)receptors. In the oxytocin and vasopressin receptors, thisposition is occupied by Gln [Gln3.32(119), Gln3.32(131)], which isalso important for ligand binding (178). Asn5.39(212) is repre-sented by important Thr residues in position 5.39 in themuscarinic receptors and Val or Ala in the adrenergic re-ceptors. Trp6.48(280) is conserved and shown to be crucial forfunction of rhodopsin, muscarinic, dopamine, serotonin,TRH, and angiotensin receptors. The equivalent residue toAsp7.32(302) in EC3 of the human GnRH receptor is con-served with the AT1-angiotensin II receptor. In both the AT1-angiotensin II- and GnRH receptors, this residue at the endof EC3 is important for high-affinity peptide-agonist binding.TRH binding also appears to involve an initial interaction

with EC3 to tether it for subsequent interaction with thedeeper binding pocket in a similar way to GnRH (179). EC3therefore appears to have become an important interactionfor small peptides (3–10 amino acids) as an adjunct to theinteractions with TM residues similar to those used by bio-genic amines.

VI. Receptor Activation

The molecular mechanisms underlying ligand-medi-ated receptor activation are most comprehensively under-stood for rhodopsin and are only partially elucidated fora few other GPCRs. The propagation of ligand binding bythe receptor to the signal transduction pathway within thecell involves a change in receptor conformation (180). Inthe classical De Lean model of GPCR function, the activeconformation is envisaged as a ternary complex consistingof hormone, receptor, and G protein. The model involvesan initial binding step common to both agonists and an-tagonists, followed by a transition step, exclusive to ago-nists, which leads to formation of the ternary complex. Thelater extended ternary complex model (169) also allows forspontaneous formation of a receptor-G protein complex,which has a higher affinity for agonist ligands and isstabilized by binding of agonists. Upon agonist activationof the receptor (R*), GDP is exchanged for GTP in theheterotrimeric G protein complex. Subsequent dissocia-tion of G� from G�� subunits results in GTP hydrolysis,reformation of the G�-GDP�� complex, and a return of thereceptor to the inactive (R) state (181). The Samama-revised model proposes that receptors fluctuate betweenan inactive R conformation and an active R* conformation(169). The R* conformation has high affinity for agonistsand productively stimulates G protein turnover. By sta-bilizing the R* conformation, agonists shift the equilibriumfurther to R* conformers. The models are essentially thesame in that they both require conformational change inthe receptor, one ligand-induced (conformation induction)and the other ligand-stabilized (conformation selection).Kenakin (182) proposed that GPCRs may occur in multiple

TABLE 4. Residues comprising the ligand binding pocket of GnRH receptor and other rhodopsin family G protein-coupled receptors

Rhodopsin mACh Adrenergic Dopamine Serotonin TRH Ang. II(AT1) Oxytocin Vasopressin

(V1a) GnRH

2.61 Thr94 Val91 (D2) Gln108 Asp98Phe91 (D4)

2.64 Phe86 (�1AA) Thr134 (5HT2A) Trp101His 93 (�2)

2.65 Asn1023.32 Ala117 Asp105 (M1) Asp125 (�1AB) Asp114 (D2) Asp116 (5HT1A) Gln119 Gln131 Lys121

Asp103 (M2) Asp113 (�2) Asp155 (5HT2A)Asp106 (5HT6)

5.39 Thr189 (M1) Val185 (�1AA) Asn212Thr187 (M2) Ala204 (�1AB)Thr231 (M3) Val197 (�2AA)

6.48 Trp265 Trp400 (M2) Trp386 (D2) Trp327 (5HT1B) Trp279 Trp253 Trp280Trp503 (M3) Trp336 (5HT2A)

6.60 Tyr2907.32 Asp281 Asp302

Data are extracted from the literature reviewed by Palczewski et al. (130), Shi and Javitch (294), Gershengorn and Osman (179), de Gasparoet al. (295), Yamano et al. (212), Barberis et al. (178), Gimpl and Fahrenholz (296), and van Rhee and Jacobson (297).


conformations, and the equilibria are controlled by theinteractions of strategically key residues (183). Agonistbinding may select and stabilize a particular conformation,directing a specific downstream signaling pathway (184).This has been called agonist-induced signal trafficking.Perhaps a more appropriate term is ligand-induced signalselectivity or LISS, because both agonists and classicalantagonists can selectively signal. For example, we haveshown that certain antagonists for the Gq pathway (ino-sitol phosphate production) at the type I GnRH receptorcan act as agonists on the Gi pathway as measured by theircapacity to inhibit forskolin-mediated cAMP accumula-tion in various reproductive tissue backgrounds (Ref. 185,and L. Davidson, manuscript in preparation). Similarly,with respect to ligand-specific signaling events, we havealso found that GnRH I activates Src whereas GnRH II isinhibitory on Src at the type I GnRH receptor (S. R. Maud-sley, unpublished observations).

The mechanism of agonist-induced receptor activation isthought to result from a disruption of the intramolecularconstraint networks that stabilize the ground state of thereceptor. The disruption of subsequent replacement by a newset of contacts results in the stabilization of the active con-formation of the receptor allowing binding of signal-medi-ating proteins to intracellular domains of the receptor. Valu-able information about conformational change associatedwith receptor activation emerged from the pioneering site-directed double spin labeling studies of rhodopsin (186).These indicated that receptor activation involves rotations ofthe TM helices and outward movements of the endofacialparts of TMs 2, 3, 6, and 7. These helices are packed tightlyin the ground state but open up in the activated state. Thepredominant movement of the TM helices is an approximate30° clockwise rotation (viewed from the cytoplasmic surface)of the endofacial part of TM6 relative to TM3 (187). Thisaccomplishes an 8 Å outward movement of TM6 toward theintracellular end of TM5 (186). The closure of the intracellular endsof TM5 and 6 during receptor activation was further confirmed byagonist-induced double-cysteine cross-linking in the M3 musca-rinic acetylcholine (188). A small outward movement (about 2–4Å) of TM2 toward TM4, and TM7 toward TM6 also occurs duringphotoactivation of rhodopsin (186). There is probably a smallmovement and rotation of TM3, which accompanies the move-ment of TM2, whose endofacial parts may act as a unit (149, 189).Inhibition of the movement between TM3 and TM6 by cross-linking blocks receptor activation (190, 191). As a consequence,TM3 becomes less tilted, and its intracellular end may be rear-ranged into the middle of TM2 and TM4. This brings TM3 moreperpendicular to the plane of the membrane and closer to TM7,thereby eliminating cavities at the intracellular end of TM3. Evi-dence of the movement of TM3 also emerged in a recent cross-linking experiment in which a photoreactive retinal analog labeledTM6 in the dark state as the crystal structure predicts, but reactedwith TM4 after light activation (192). In the crystal structure, amodeled all-trans retinal conformation passes through TM3, andtherefore TM3 must be nudged away from this area to open a waybetween TM7 and TM4 (191). The reconfiguring of TM3 is alsoconsistent with the presence of cavities at the intracellular end ofTM3.

A. Interaction of Asn2.50(87)/Asp7.49(319) in TM2/7 in GnRHreceptor activation

Mutation of Asn2.50(87) and Asp7.49(318) in the mouse GnRHreceptor [Asn2.50(87) and Asp7.49(319) in the human] (Figs. 7and 8) revealed that Asp7.49(318) is involved in receptor acti-vation (signal propagation) because the mutants Asn2.50(87)

Asn7.49(318) and Asp2.50(87)Asn7.49(318) both retained goodligand binding but poor stimulation of inositol phosphateproduction (140). These findings indicate that the unusualarrangement of having Asp7.49 in TM7 in the GnRH receptoris an essential component of ligand-mediated receptor acti-vation, which normally is subserved by the conserved Asp2.50

in TM2 of other GPCRs and the nonmammalian GnRH re-ceptors (121) (Fig. 8).

Interestingly, the presence of an Asp7.49 in TM7 of theGnRH receptor facilitates coupling to phospholipase C (PLC)via Gq/11 but prevents coupling to phospholipase D (PLD) bythe small monomeric G protein (147). Mutation to Asn7.49,which is present in the majority of GPCRs, recreates thiscoupling to PLD. Thus, the reverse arrangement of Asn2.50

and Asp7.49 in TM2 and TM7 in the GnRH receptor appearsto have been selected to allow PLC coupling and preventPLD coupling (147). Further exploration by mutation of TM2Asn2.50 and TM7 Asp7.49 to various amino acids has con-firmed that the TM2 Asn2.50 is essential for configuring andexpression of the receptor, whereas the TM7 Asp7.49 is onlyessential for receptor activation (143). Thus, these early stud-ies revealed that Asn2.50 in TM2 and Asp7.49 in TM7 are anelement of the molecular switch of receptor activation.

B. Disruption of TM3 Asp3.49(138)/Arg3.50(139) interaction inGnRH receptor activation

The highly conserved motif DRxxxI/V at the intracellularend of TM3 is also implicated in GnRH receptor activation(193, 194). In the GnRH receptor molecular model, Asp3.49(138)

and Arg350(139) (DR) (Fig. 14, A and B) appear capable of acharge interaction (193), and this has been confirmed in thecrystal structure of rhodopsin (130). Disruption of this bymutating Asp to an uncharged residue in the GnRH receptorconveys increased coupling efficiency, possibly through therelease of Arg3.50(139) to interact with other residues (193).This suggests that the Asp3.49(138)Arg350(139) charge interac-tion is disrupted in the active state of the receptor. TheIle3.54(143) located one helical turn below the Arg350(139) ap-pears to play a role in caging the Arg3.50(139) side chain forcoupling interactions by sterically limiting its movement.Mutation to small residues (e.g., Ala) results in some uncou-pling (193). It is proposed that the Arg3.50(139) side chain isinvolved in a triad interaction with the TM2 Asn2.50 and TM7Asp7.49 in stabilizing the active conformation of the receptor(193) (Fig. 14B). The Arg3.50(139) is crucial for coupling becausemutation to Gln leads to very poor coupling efficiency (193).The receptor activation-induced movements of TM helicesdescribed above may lead to the Arg3.50(139) side chain in-teracting with Tyr7.53(323) in the conserved TM7 N/DPxxYmotif in the active conformation, which was proposed byVriend and colleagues (195). This highly conserved Tyr7.53 iscritically important for G protein signaling in the GnRH


receptor (144) and in other rhodopsin family GPCRs(196–201).

Both Arg3.50(139) and Tyr7.53(323) can make multiple in-tramolecular contacts, such as H-bond, hydrophobic, andcationic-� interactions, which may account for the distinctiveresults caused by mutagenesis of both residues. Thus, anessential element of activation of the GnRH receptor andother GPCRs appears to be an agonist-induced (or stabilized)disruption of intramolecular constraint networks accompa-nied by the protonation of Asp3.49(138) to release Arg3.50(139)

for interaction with the Asn2.50(87)/Asp7.49(319) residues inTM2 and TM7 and possibly Tyr7.53(323). Because the con-served Asn1.50 in TM1 of rhodopsin interacts with Asp2.50 inTM2 in the inactive state (130), this residue may also play arole in the TM domain network involved in receptor acti-vation. We have been unable to explore this because muta-tion of Asn1.50(53) leads to an absence of detectable bindingand signaling, which may be due to nonfunctional receptoror poor binding.

C. The triad of Glu2.53(90)-Lys3.32(121)-Asp2.61(98)

Flanagan et al. (159) have produced experimental evidencesuggesting an interaction of Asp2.61(98) with Lys3.32(121). Stud-ies on a GnRH receptor model have proposed that an H-bondnetwork of Glu2.53(90)-Lys3.32(121)-Asp2.61(98) is present in theinactive state (Figs. 13B and 14A) and is replaced by a newset of intermolecular contacts between Lys3.32(121)-His2(GnRH)-Asp2.61(98) in the active ligand-bound state (146)(Fig. 13A). This change may form part of the molecularswitch in agonist activation of the receptor (146). This hy-pothesis is supported by the demonstration that Lys3.32(121) isessential for agonist binding but not antagonist binding (160)and the observation of a 100-fold decrease in affinity of nativeGnRH (His2) binding by the Asp2.61(98)Glu mutant and main-tenance of mutant binding affinity for Trp2 GnRH (159).Glu2.53(90) mutation to Gln had no effect on function (68), butmutation to Ala resulted in complete loss of function (162).Presumably Gln2.53(90) is able to maintain hydrogen bondingwith Lys3.32(121).

D. Role of extracellular loop 2

The disulfide bridge between the extracellular end ofTM3 and EC2 is conserved in all of the GPCRs in therhodopsin family and is essential for receptor function(202). Because Lys3.32(121) and Asp3.49(138)/Arg3.50(139) inTM3 play a major role in receptor activation, the rigidconnection of TM3 to EC2 through the disulfide bridgesuggests an important role of this extracellular domain inthe assumption of the active and inactive states of thereceptor. This supposition is supported by reports thatantibodies against EC2 of the �1 (203), �1- and �2-adren-ergic (204), AT1-angiotensin II (205), bradykinin B2 (206),and M1- (207) and M2-muscarinic-acetylcholine (208) re-ceptors can activate second messenger responses, presum-ably by stabilizing the receptor in the active conformation.Changes in the EC2 configuration might translate intostabilizing TM3, TM4, and TM5 in the active conformationdue to its physical connection to these domains. The crys-

FIG. 14. Proposed intramolecular interactions associated with theactive and inactive states of the human GnRH receptor. A, Spatialpositioning of residues involved in intramolecular interactions in theinactive state of the human GnRH receptor. B, A three-dimensionalmodel of the TM domains (TM2, TM3, and TM7) shows how theprotonation of Asp3.49(138) breaks the ionic bond with Arg3.50(139) tofacilitate hydrogen bond formation with Asn2.50(87) and Asp7.49(319) inthe active conformation of the human GnRH receptor (193).


tal structure of rhodopsin reveals that EC2 has a distinctstructure intimately associated with the TM domains.

Strong evidence has been obtained for a role of EC2 in theactivation of the human GnRH receptor (209). Certain an-tagonists at the human GnRH receptor are agonists at thechicken (71) and Xenopus (209) receptors. Similarly, incor-poration of chicken receptor EC domains into the humanreceptor established that this domain is responsible for confer-ring agonist activity to a GnRH antagonist (71). By incorporat-ing the Xenopus GnRH receptor extracellular domains into thehuman GnRH receptor, Ott et al. (209) demonstrated that theXenopus EC2 could convey agonism to GnRH antagonist 135–18. Mutation of amino acids in the human GnRH receptor EC2,which differed in the Xenopus EC2, revealed that a minimum ofVal5.26(197)Ala together with Trp5.32(205)His was sufficient to con-vey agonism to antagonist 135–18. By comparing agonistic be-havior of a range of GnRH antagonists, a single residue d-Lys(iPr) in position 6 was shown to be responsible for thephenomenon (209). Together with the pH dependence of theeffect, the findings suggested that His5.32(205) forms a charge-supported hydrogen bond with d-Lys(iPr)6 of the antagonist tostabilize the receptor in the active conformation. The recentdemonstration that a single mutation of Ala5.25(201) to Thr in EC2

in combination with a TM6 mutation in the frog GnRH receptoralters signaling further underlines the importance of EC2 inreceptor activation (115).

E. Other residues possibly involved in receptor activation

Other potential elements of GnRH receptor activationmay include the interactions between Asn5.39(212) of TM5and pGlu1 of GnRH, and between Tyr6.58(290) and Tyr5 ofGnRH (162, 163). In another GnRH receptor model, Trp (3)of GnRH was predicted to penetrate about 20 Å into theTM core and interact with Trp6.48(279) [Trp6.48(280) in thehuman receptor] of TM6 (164) and potentially intercalatebetween the indole moiety of Trp6.48(279) and the phenylmoiety of Phe7.41(310) [Phe7.41(311) in the human receptor] inTM7 (166). Mutation of Trp6.48(279) to Ser or Phe7.41(310) toLeu (present in most GPCRs) reduced binding affinityslightly but totally abrogated coupling to inositol phos-phate production. Because mutation of both residues to-gether was not additive, it was interpreted that they in-teract and constitute part of the activation network. Trp6.48

in other GPCRs (e.g., rhodopsin, cholecystokinin B, and

FIG. 15. A schematic model showing the interactions of GnRH NH2-terminal contact sites (pGlu1, His2, Trp3) and intramolecular interactionsof the human GnRH receptor in the proposed active conformation (red residues and lines) compared with the intramolecular interactions inthe inactive conformation (black lines). GnRH residues involved in binding but not activation are shown in black. Experimental data supportthe concept that Asp3.49(138) and Arg3.50(139) form an ionic bond in the inactive conformation of the receptor and that this is broken by protonationof Asp3.49(138) in the active conformation of the receptor (193). This allows interaction of Arg3.50(139) with Asn2.50(87) and Asp7.49(319), and possiblyTyr7.53(323). These new interactions potentially cause changes in the orientation of TM3 and TM7 �-helices, which in turn propagate into changesin the other helices [e.g., TM4 and TM5 through the disulfide bridge (Ref. 148) and EC3]. Soderhall et al. (146) have proposed that His2 of GnRHdisrupts a hydrogen bond triad of Glu2.53(90)/Lys3.32(121)/Asp2.61(98) in the active state of the receptor. In this event, a parsimonious proposal isthat in the active state of the receptor, protonation of Asp3.49(138) in TM3 is accompanied by a disruption of the Glu2.53(90)/Lys3.32(121)/Asp2.61(98)

hydrogen bond triad that is stabilized by the interaction of His2 of GnRH with Lys3.32(121) and Asp2.61(98).


angiotensin AT1A receptors) plays a crucial role in recep-tor function (210 –212).

F. Integrated model of GnRH receptor activation

The data described above point to a number of intramo-lecular changes associated with the interconversion of activeand inactive configurations of the human GnRH receptor.Precisely how these changes relate to each other in receptoractivation is currently conjectural. We propose that in theinactive state Asp3.49(138) forms a charge interaction withArg3.50(139), Glu2.53(90)-Lys3.32(121)-Asp2.61(98) form a hydrogenbond network, and Asn2.50(87) interacts with Asn1.50(53) andAsp7.49(319) (Fig. 15). In the active configuration, Asp3.49(138) isprotonated, releasing Arg3.50(139) to interact with Asn2.50(87),Asp7.49(319), and possibly Tyr7.53(323) (Fig. 15). Asn2.50(87) mayalso interact with Asn1.50(53) in TM1 in the active conforma-tion because this residue is crucial for receptor function.These new interactions may be accompanied by a loss ofinteraction of the Glu2.53(90)-Lys3.32(121)-Asp2.61(98) triad, al-lowing the binding of GnRH and the formation of the contactLys3.32(121)-His2(GnRH)-Asp2.61(98). These changes give rise toa rotation of TM3, which affects the other TMs directly (e.g.,TM4 and TM5 through the disulfide bridge between TM3and EC2) and indirectly through interhelical interactions[e.g., TM2 and TM7 through Asn2.50(87) and Asp7.49(319) in-teractions]. These helical movements are thought to be as-sociated with a different configuration of the connected ICs,which favors binding/activation of intracellular signalingproteins.

In contrast to the mammalian type I GnRH receptor, themolecular mechanisms of activation of nonmammalianGnRH receptors have received much less attention. All of theresidues identified in the activation of mammalian receptorsare present in nonmammalian receptors except for the sub-stitution of Asn2.50(87) with Asp to give rise to Asp in bothTM2 and TM7 (Fig. 8), which is present in a number of

GPCRs. Like the majority of GPCRs, and contrary to themammalian GnRH receptor, the Asp2.50 in TM2, and not theAsp7.49 in TM7, is crucial for receptor binding and activation(121). This finding is therefore similar to that in other GPCRs(e.g., 5HT2A) and opposite to that observed in mammaliantype I GnRH receptors in which the Asp7.49 in TM7 is es-sential for coupling. Interestingly, the total loss of function ofthe catfish receptor TM2 Asp2.50(90)Asn mutant can be par-tially restored by mutation of a Met2.53(93) one helical turnabove to Glu as in the mammalian GnRH receptor [Glu2.53(90),Fig. 8] (168). It appears therefore that the unusual reciprocalarrangement of these interacting TM2/TM7 residues in themammalian receptor is related to the loss of the carboxyl-terminal tail. We propose that the efficient functioning of themammalian type I GnRH receptor required the loss of thecarboxyl-terminal tail such that it did not undergo rapiddesensitization (see Section VIII.C). The loss of the tail becamethe driving force for coordinated molecular changes to thereceptor.

VII. GnRH Receptor Mutations in HypogonadotropicHypogonadism

Fourteen mutations of the GnRH receptor have been de-scribed in hypogonadotropic hypogonadism (213–222) (Ta-ble 5). These patients are characterized by delayed sexualdevelopment and inappropriately low or apulsatile gonad-otropin and sex steroid hormone levels in the absence offunctional abnormalities of the hypothalamopituitary axis(223). Although some of the mutants are totally nonfunc-tional in vitro [Glu2.53(90)Lys, Ala3.40(129)Asp, Arg3.50(139)His,Ser4.54(168)Arg, Cys5.27(200)Tyr, Ser5.44(217)Arg, Leu6.34(266)Arg,Cys6.47(279)Tyr, and a truncation at Leu7.44(314)], others havesome ability to elicit an inositol phosphate response to GnRH[Asn(10)Lys, Thr(32)Ile, Gln2.69(106)Arg, Arg6.30(262)Gln, andTyr6.52(284)Cys] (see Table 3 for references).

TABLE 5. Characteristics of natural GnRH receptor mutations

Mutation Site Ligand binding(vs. WT)

Maximal IP production(vs. WT)

Cell surface expressionassessed with:

Asn(10)Lys NH2-terminal domain �� to ��/�� Normal GFPThr(32)Ile TM1 NE 0 HAGlu2.53(90)Lys TM2 Not detectable 0 NEGln2.69(106)Arg EC1 �/�� 0 to ��/�� HAAla3.40(129)Asp TM3 Not detectable 0 NEArg3.50(139)His TM3 Not detectable 0 GFPSer4.58(168)Arg TM4 Not detectable 0 GFPAla4.57(171)Thr TM4 0 0 GFPCys5.27(200)Tyr EC2 NE 0 HASer5.44(217)Arg TM5 Not detectable 0 NEArg6.30(262)Gln IC3 ��/�� to Normal �� to ��/�� NELeu6.34(266)Arg TM6 NE 0 HACys6.47(279)Tyr TM6 NE 0 HATyr6.52(284)Cys TM6 �/�� /�� NELeu7.44(314)X TM7 Not detectable 0 NE

IP, Inositol phosphates; NE, not evaluated; HA, hemophillus antigen; GFP, green fluorescent protein; WT, wild type. (Genotype)-phenotype:[Gln2.69(106)Arg/Gln2.69(106)Arg]-partial, [Gln2.69(106)Arg/Arg6.30(262)Gln]-partial, [Gln2.69(106)Arg-Ser217Arg/Arg6.30(262)Gln]-complete to partial,[Gln2.69(106)Arg/Leu7.44(314)Xstop]-complete, [Gln2.69(106)Arg/Ala4.57(171)Thr]-complete, [Gln2.69(106)Arg/Leu6.34(266)Arg]-partial, [Gln2.69(106)Arg-Ser5.44(217)Arg/Asn10Lys]-partial, [Arg6.30(262)Gln/Tyr6.52(284)Cys]-not reported, [Arg6.30(262)Gln/Ala3.40(129)Asp]-complete, [Ser4.58(168)Arg/Ser4.58(168)Arg]-complete, [Thr32Ile/Cys5.27(200)Tyr]-complete, [Cys6.47(279)Tyr/Cys6.47(279)Tyr]-complete, [Glu2.53(90)Lys/Glu2.53(90)Lys]-complete,and [Arg3.50(139)His/Arg3.50(139)His]-complete. [Adapted with permission from A. Leanos-Miranda et al.: J Clin Endocrinol Metab 87:4825–4828,2002 (167). © The Endocrine Society.]


Six of the mutations are at sites previously identified inmutagenesis studies as important for receptor function.Glu2.53(90)Ala (162) and Arg3.50(139)His (160) were totally in-active as in the corresponding patient mutants. BothGlu2.53(90) and Arg3.50(139) residues are thought to be involvedin receptor activation. On the other hand, Cys5.27(200)Ser dif-fered from the inactive patient mutant of Cys5.27(200)Tyr inhaving reduced affinity and inositol phosphate production(148). Ser is a homolog of Cys with an oxygen atom substi-tuting for sulfur. The large aromatic Tyr is thus likely toproduce a more deficient phenotype. Conversely, Ser5.44(217)

mutation to Ala had no effect on receptor function in vitro(162) in contrast to the total absence of activity for the patientmutant Ser5.44(217)Arg. This suggests that Ser5.44(217) in TM5does not serve an important role and can be substituted witha small residue (Ala), but a large positively charged residue(Arg) disrupts function. Mutation of Arg6.30(262) to Lysproduced a partially active receptor (21), as did theArg6.30(262)Gln patient mutant, suggesting H bonding capac-ity. Tyr6.52(284) mutation to Cys in a patient produced a par-tially active receptor. Mutation of Tyr6.52(284) to Ala was re-ported to result in an inactive receptor (163), but we havefound that this mutation results in poor expression and lowaffinity. These naturally occurring mutations thus shed fur-ther light on the molecular functioning of the human GnRHreceptor and provide insight for additional experimentation.

Recently, a cell permeant small molecule antagonist (com-pound 11 in Fig. 6) was shown to rescue all of the naturallyoccurring mutants except Ser4.54(168)Arg, Ser5.44(217)Arg, andLeu7.44(314)Stop by increasing their expression (167). Thesefindings suggest that the majority of mutations result in aninstability or misfolding of the GnRH receptor and that thesmall molecule antagonist stabilizes these mutants and pro-tects them from targeting to degradative pathways in theendoplasmic reticulum (224). These intriguing observationsoffer the possibility of using cell-permeant small moleculeantagonists as therapeutics for GnRH receptor and otherGPCR mutations, and also as a valuable tool for character-izing the properties of poorly expressed experimentalmutants.

VIII. Structural Correlates of GnRH ReceptorCoupling and Internalization

GnRH agonist occupancy of GnRH receptors leads to ac-tivation of multiple signal transduction pathways. In gona-dotropes, GnRH activates PLC� via Gq/11�, resulting in thehydrolysis of membrane-bound phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-triphosphate and diacylglyc-erol, which mobilize intracellular calcium and activate PKC,respectively. These in turn stimulate the biosynthesis andsecretion of the gonadotropins, LH and FSH. These intra-cellular signaling pathways have been reviewed extensively(27, 29, 30, 225–227, 227a) and will not be discussed furtherhere. Instead, in this section we focus our attention on thestructural features of GnRH receptors that determine theircoupling to different intracellular proteins mediating intra-cellular signaling and internalization of the receptors. Ad-ditionally, we consider the new concept that different GnRH

ligands can determine preferential interactions with differentintracellular proteins through stabilization of the GnRH re-ceptor in different conformations.

Our recent unpublished observations have led us to for-mulate a novel concept which proposes that the GnRH re-ceptor can assume a number of different active conforma-tions that preferentially and selectively activate differentintracellular signalling pathways (R. P. Millar, Z.-L. Lu, A. J.Pawson, C. A. Flanagan, K. Morgan, and S. R. Maudsley,unpublished observations). Furthermore, the different activeconformations are stabilized (activated) by different GnRHanalogs. The challenge now will be to determine the natureof these specific ligand-receptor interactions and receptorconformations that transduce specific signalling (which wehave termed GnRH ligand-selective signalling) and physi-ological and pathophysiological effects.

A. Coupling to multiple G proteins

Gq/11 is the predominant G protein coupled to the GnRHreceptor in various cellular environments (27, 225, 228). Anumber of studies have demonstrated that other G proteinscan mediate the actions of GnRH receptors. Pretreatment ofrat pituitary cells with pertussis toxin decreased inositolphosphate production in response to GnRH, suggesting cou-pling to either Gi or Go (229). In addition, GnRH receptorcoupling to Gi has been demonstrated in ovarian carcinomas(230–232), uterine leiomyosarcomas (230), uterine endome-trial carcinomas (232, 233), and human prostate cancer cells(234). Pretreatment of rat pituitary cells with cholera toxinresults in an increase in GnRH stimulation of LH, suggestingcoupling to Gs (235, 236). In addition, Gs and Gi coupling hadbeen revealed by the GnRH stimulation of cAMP in a numberof experimental paradigms (226, 237–241). What are thestructural features of the GnRH receptor that facilitate thesedifferences in coupling?

The ICs and carboxyl-terminal tail have been implicatedin specific coupling of GPCRs to G proteins, but theirdegree of involvement varies among different receptors.Because all the mammalian GnRH receptors lack a car-boxyl-terminal tail, effective receptor-G protein interac-tions must take place via one or more of the ICs. Theconservation of the carboxyl-terminal sequence of IC3 invertebrate GnRH receptors (Fig. 8) suggested that thisregion may be crucial for coupling to the primary medi-ator, Gq/11. A series of cassette substitutions covering theentire sequence of IC3 confirmed this hypothesis (I. Wake-field, unpublished observations). Within this region,Ala6.29(261) was identified as an important residue for cou-pling. When the equivalent Ala in IC3 of biogenic aminereceptors is mutated to large residues, the receptors areconstitutively active (242). However, mutation ofAla6.29(261) to bulky amino acids resulted in an oppositeeffect in the GnRH receptor, namely uncoupling of thereceptor and failure to generate inositol phosphate (243).Mutation of the evolutionarily conserved adjacent basicamino acid [Arg6.30(262)] to Ala was also shown to result inuncoupling (I. Wakefield, unpublished observations), andnatural mutations of Arg6.30(262) have been shown to causeuncoupling in receptors of families with hypogonadotro-


phic hypogonadism (213, 216, 244). The effects of overex-pression of rat GnRH receptor IC3 peptides in GnRH re-ceptor-expressing GGH3 cells on inositol phosphateproduction and cAMP accumulation demonstrated thatthis domain is involved in coupling to Gq/11 and Gs signaltransduction pathways (245).

The GnRH receptors have the conserved motif DRxxxI/VxxPL at the N terminus of IC2, which plays a role in cou-pling. The importance of the DRxxxI element in receptoractivation (see Section VI) appears to extend to thePro3.57(146)Leu3.58(147). Mutation of the preceding Arg3.56(145)

to Pro causes uncoupling (246), presumably because thismutation introduces a Pro-Pro motif known to disrupt sec-ondary structure. Replacement of the conserved Leu3.58(147)

with Asp or Ala led to defective Gq/11 coupling (247), andmutation of Arg3.50(139) of the mouse GnRH receptor to Glnproduced a similar effect (194).

In IC1, the sequence (KKLSR) is a Gs recognition motif(BBxxB, where B is a basic amino acid); mutation of certainof these residues leads to uncoupling of cAMP productionbut not of inositol phosphate production (239).

The above studies indicate that the GnRH receptor is ableto couple to several G proteins and activate a number ofeffectors via different elements of the three ICs. It appearsthat coupling to Gq/11 occurs through IC2 and IC3, and to Gsthrough IC1. Coupling to Gi is less understood, but may berelated to the cell-type, stage of the cell cycle, or availabilityof the Gi protein. Recently, it has been suggested that Giactivation underlies the antiproliferative effects of GnRH inmany cancers (231, 248). The IC elements for Gi couplinghave not been investigated.

The carboxyl-terminal tail of GPCRs has also been implicatedin the regulation of signaling via receptor-coupled G proteins.In contrast to the mammalian GnRH receptors, but in commonwith other GPCRs, the cloned nonmammalian GnRH receptorsall have a carboxyl-terminal tail (18, 51, 65, 67, 71). The absenceof a carboxyl-terminal tail in mammalian GnRH receptors iscorrelated with a lack of rapid desensitization (249), in contrastto nonmammalian and type II tailed receptors that exhibit rapiddesensitization. A number of studies (250–254) involving trun-cation, site-directed mutagenesis, and carboxyl-terminal tail-swapping have established the importance of this region forcoupling, desensitization (reviewed in Ref. 255), and receptorinternalization (see Section VIII.C). Although mammalian re-ceptors lack the carboxyl tail, the carboxyl-terminal residues ofTM7 are important for Gq/11 effector coupling (256). Because thelast four residues (YFSL) of all mammalian GnRH receptors area conserved putative class II postsynaptic density, discs-large,ZO-1 (PDZ) domain-binding motif, these residues may be im-portant for effective mammalian GnRH receptor signaltransduction.

B. Regulators of G protein signaling (RGS) proteins

Inactive G proteins are heterotrimers consisting of the �-,�-, and �-subunits. Upon receptor activation, GTP displacesGDP from binding to the G�-subunit and is then hydrolyzedto GDP by intrinsic GTPase activity. This promotes the re-association of G�-subunit and ��-dimers, forming the inac-tive heterotrimer. RGS proteins interact directly with active

G�-subunits to accelerate their intrinsic GTPase activity andlimit their half-life (257). Two family members, RGS3 andRGS10, have been implicated in the regulation of GnRHreceptor coupling (258–260). Furthermore, there is evidencethat the carboxyl-terminal tails of nonmammalian GnRHreceptors may be sites for interactions with RGS10, althoughthe nature of this interaction is unclear (260).

In addition to RGS proteins, there is scope and precedencefor involvement of accessory proteins such as arrestins,GPCR kinases, Src-homology domain 2 domain-containingproteins, small GTP-binding proteins (261), polyproline-binding proteins, receptor-activity-modifying proteins, andmembers of the scaffolding family of proteins such as PDZdomain-containing proteins (262) in the regulation GnRHreceptor coupling and signal transduction. The requisite se-quence structural motifs within the GnRH receptors have notbeen identified.

C. GnRH receptor internalization

At least four pathways of agonist-induced internalizationof GPCRs exist (263), which may be cell-type specific. Theclassical GPCR internalization pathway involves GPCR ki-nases, �-arrestin, clathrin-coated pits, and the GTPase dy-namin, and is exemplified by the �2-adrenergic receptor(263–268). Upon receptor activation, G protein-coupled re-ceptor kinases are targeted to the receptor by the generationof free ��-subunits. Activated G protein-coupled receptorkinases phosphorylate the receptor at specific serine andthreonine residues. Receptor phosphorylation enhances thebinding of �-arrestin. �-Arrestin binding interdicts G proteincoupling and also serves to target the receptor to clathrin-coated pits for internalization. Dynamin is thought to benecessary for the scission of clathrin-coated vesicles from theplasma membrane (269). GPCRs have also been reported tointernalize independently of both �-arrestin and dynamin, orin pathways dependent on only one or the other, implyingthat GPCRs are able to undergo internalization via pathwaysthat are distinct from clathrin-coated pits (263–267). The sub-cellular localization of certain GPCRs to smooth noncoatedmembrane structures and vesicles (270, 271) suggests thatGPCRs can use internalization pathways that are distinctfrom clathrin-coated vesicles. Caveolae are flask-shaped,nonclathrin-coated structures that have been implicated inthe internalization of small molecules and certain GPCRs(272–277). In addition, dynamin has been implicated incaveolae function, although its exact functional role is notknown. As for clathrin-coated pits, dynamin may be respon-sible for the pinching off of caveolae from the plasma mem-brane (278, 279).

The internalization pathways used by GnRH receptors arecell-type dependent and also differ between different receptorsubtypes. The rat GnRH receptor, which lacks a cytoplasmictail, internalizes in a �-arrestin-independent manner, but prob-ably via a clathrin-dependent mechanism (250), and in a �-ar-restin-independent pathway that is dynamin-dependent (253).The lack of a carboxyl-terminal domain in the mammalianGnRH receptors probably accounts for their �-arrestin inde-pendency for internalization. A comparison of the pathways ofinternalization of the human and Xenopus type I GnRH recep-


tors in HeLa cells reported that the human GnRH receptorinternalizes in a dynamin-independent manner, whereas theXenopus type I GnRH receptor internalizes in a dynamin-de-pendent manner (176). Despite the differences, both appear tointernalize via a pathway that is clathrin-mediated (176). Sim-ilarly, rat GnRH receptors colocalize with transferrin receptorsthat are known to internalize in clathrin-coated vesicles (250).Based on the above studies, it appears that mammalian andnonmammalian GnRH receptors can both be targeted for clath-rin-mediated internalization.

The carboxyl-terminal tail of the catfish GnRH receptor isimportant for cell surface expression, ligand binding, andreceptor phosphorylation and internalization (121, 252).Agonist-induced internalization of the catfish GnRH recep-tor (252) is dependent on a serine residue in the carboxyl-terminal tail that is phosphorylated and may function as a�-arrestin binding site. Addition of the carboxyl-terminal tailof the catfish GnRH receptor and TSH-releasing hormonereceptor to the rat GnRH receptor results in an increased rateof internalization (280, 281).

The chicken GnRH receptor (71) exhibits rapid internal-ization kinetics and was shown to be dependent on the car-boxyl-terminal tail for this process (251). A threonine-doublet(Thr369Thr370) located at the distal end of the cytoplasmic tailis critical, because its mutation to Ala residues abolished rapidinternalization (254). The chicken GnRH receptor preferentiallyundergoes rapid agonist-induced internalization in a dynamin-and caveolae-dependent manner, based on the finding thatinternalization is inhibited in the presence of dominant negative(K44A) dynamin-1 and caveolin-1(�1–81) overexpression, andpretreatment with the caveolae disruptors, filipin and methyl-�-cyclodextrin (254). However, internalization of the chickenGnRH receptor in COS-7 cells was independent of �-arrestinand clathrin-coated vesicles (254).

A recent study has characterized the internalization path-ways of the three bullfrog GnRH receptor subtypes (282). Thebullfrog type II GnRH receptor (reclassified as bullfrog typeI here; see Fig. 8) showed the most rapid rate and highestextent of internalization among the three receptors. Further-more, internalization of the bullfrog type I GnRH receptorwas shown to be both �-arrestin and dynamin-dependent,whereas bullfrog type II and III GnRH receptors internalizevia a pathway that is �-arrestin-independent, but dynamin-dependent, similar to the pathway used by the chickenGnRH receptor (254, 282). It is interesting to note that the lasteight residues of the carboxyl-terminal tails of the chickenand bullfrog type II GnRH receptors are surprisingly similar(GTTVNTVC for chicken, ATTVQSVF for bullfrog type II).This may point to the importance of the Thr-doublet that wasidentified as critical for chicken GnRH receptor internaliza-tion (254). The above studies thus suggest that the carboxyl-terminal tail of the nonmammalian GnRH receptors plays apivotal role in their function and subcellular trafficking.

IX. Conclusions and Future Perspectives

The fundamental role of hypothalamic GnRH in the re-productive system through stimulating pituitary gonadotro-pin secretion has made it a prime drug target for treatment

of infertility and sex hormone-dependent diseases and fornovel contraception. It is now clear that GnRHs have beenco-opted during evolution for other functions in addition toregulating gonadotropins. The identification of structuralvariants of GnRH in extrahypothalamic tissues and the dis-covery of their cognate GnRH receptor types are providingconsiderable insight into novel physiological and pathophys-iological roles of GnRHs in diverse processes. A detailedmolecular delineation of the interaction of these GnRHs withthe type I GnRH receptor and the selective activation ofintracellular signals will contribute to the development ofnovel GnRH therapeutics.

Acknowledgments

We thank Carol Adam and Ted Pinner for expert preparation of thismanuscript.

Address all correspondence and requests for reprints to: ProfessorRobert P. Millar, Medical Research Council Human ReproductiveSciences Unit, The Chancellor’s Building, 49 Little France Crescent,Edinburgh EH16 4SB, Scotland, United Kingdom. E-mail: [email protected]

This work was supported by the Medical Research Council (UnitedKingdom), a transnational grant from the Medical Research Council(South Africa), the National Research Foundation (South Africa), andThe Wellcome Trust (United Kingdom).

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AMERICAN BOARD OF INTERNAL MEDICINE

2004 Certification Examination in Endocrinology, Diabetes, and Metabolism

Registration Period: January 1, 2004–April 1, 2004Late Registration Period: April 2, 2004–June 1, 2004

Examination Date: November 3, 2004

Important Note: The Board now offers all of its Subspecialty Certification Examinations annually.

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CPD Examination Administration Registration PeriodMay 4, 2004 December 1, 2003–March 1, 2004November 3, 2004 June 1, 2004–September 1, 2004

For more information and application forms, please contact:Registration Section Telephone: (800) 441-2246 or (215) 446-3500American Board of Internal Medicine Fax: (215) 446-3590510 Walnut Street, Suite 1700 Email: [email protected], PA 19106-3699

Note: Physicians can now register for all ABIM certification and recertification examinations via theBoard’s web site at www.abim.org (click on “Online Services”).

Endocrine Reviews is published bimonthly by The Endocrine Society (http://www.endo-society.org), the foremost professional societyserving the endocrine community.


Documents

Gonadotropin-Releasing Hormone Receptors