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Medical Research Council Human Reproductive Sciences Unit (R.P.M., Z.-L.L., A.J.P., K.M., S.R.M.), Centre for Reproductive Biology, Edinburgh EH16 4SB, Scotland, United Kingdom; and Division of Medical Biochemistry and Department of Medicine (R.P.M., C.A.F.), University of Cape Town Faculty of Health Sciences, Cape Town 7925, South Africa
Correspondence: Address all correspondence and requests for reprints to: Professor Robert P. Millar, Medical Research Council Human Reproductive Sciences Unit, The Chancellor’s Building, 49 Little France Crescent, Edinburgh EH16 4SB, Scotland, United Kingdom. E-mail: r.millar{at}hrsu.mrc.ac.uk
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Abstract |
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 Top Abstract I. IntroductionII. Structure of GnRHs...III. Structure of GnRH...IV. Binding of GnRH...V. Binding Interactions of...VI. Receptor ActivationVII. GnRH Receptor Mutations...VIII. Structural Correlates of...IX. Conclusions and Future...References |
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I. Introduction |
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TopAbstractI. IntroductionII. Structure of GnRHs...III. Structure of GnRH...IV. Binding of GnRH...V. Binding Interactions of...VI. Receptor ActivationVII. GnRH Receptor Mutations...VIII. Structural Correlates of...IX. Conclusions and Future...References |
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Low doses of synthetic GnRH delivered in a pulsatile fashion to simulate the endogenous GnRH levels in the portal vessels (picograms per milliliter) restore fertility in hypogonadal men and women and are also effective in the treatment of undescended testes and delayed puberty (6, 7, 8, 9, 10, 11, 12). However, high doses of GnRH or agonist analogs desensitize the gonadotrope with resultant decrease in LH and FSH and a decline in ovarian and testicular function (6, 7, 8, 9, 10, 11, 12, 13). This desensitization phenomenon is extensively applied in clinical medicine for the treatment of a wide range of diseases (6, 7, 8, 9, 10, 11, 12, 13) (Table 1
). GnRH peptide antagonists also inhibit the reproductive system through competition with endogenous GnRH for receptor binding, but the doses required are higher than the desensitizing agonist doses, presenting challenges for administration in the treatment of chronic diseases (14). The development of novel delivery systems for peptide antagonists or the development of nonpeptide orally active GnRH antagonists (14) is therefore likely to replace agonist therapy and avoid the undesirable stimulation and disease flare that precedes desensitization. In addition to the therapeutic applications, GnRH analogs are predicted to be used as new generation male and female contraceptives in conjunction with steroid hormone replacement (15, 16, 17).
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This article reviews our current knowledge on the structure, ligand interactions, and activation of the type I GnRH receptor. In addition, the array of novel GnRH receptors in mammals and nonmammals and their relationships and differences will be described. The review will only briefly cover current knowledge on GnRH structural variants, their possible functions, and structure-activity relations of GnRH analogs because these have been thoroughly reviewed previously and have not been subject to major new developments (18, 22, 23, 24). Nevertheless, information on the structures and subtype classification of naturally occurring GnRHs and GnRH analogs is provided because this is required for the discussion of ligand selectivity and ligand interactions of GnRH receptors. Considerable attention will be given to the molecular functioning of the GnRH receptor as new insights have recently emerged. The important area of GnRH-mediated intracellular signaling will not be covered because it has been the subject of recent comprehensive review (25, 26, 27, 28, 29, 30), but receptor structural elements involved in binding and activation of signaling proteins and internalization of receptors will be addressed in detail.
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II. Structure of GnRHs and Analogs |
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TopAbstractI. IntroductionII. Structure of GnRHs...III. Structure of GnRH...IV. Binding of GnRH...V. Binding Interactions of...VI. Receptor ActivationVII. GnRH Receptor Mutations...VIII. Structural Correlates of...IX. Conclusions and Future...References |
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). These are distributed in a wide range of tissues in vertebrates in which they apparently have diverse functions, including neuroendocrine (e.g., GH release in certain fish species), paracrine (e.g., in placenta and gonads), autocrine (e.g., GnRH neurons, immune cells, breast and prostatic cancer cells), and neurotransmitter/neuromodulatory roles in the central and peripheral nervous systems (e.g., sympathetic ganglion, mid-brain) (6, 13, 18, 22, 23, 24, 33, 34, 35, 36, 37, 38, 43, 44, 45). Because none of this signaler/target cell communication is mediated through secretion of GnRH into the general circulation, a single form of GnRH is theoretically capable of serving all of these roles (see Type II GnRH receptor, Section III.A). However, it is evident that at least two, and usually three, forms of GnRH are present in the majority of the vertebrate species studied (18, 20, 21, 22, 23, 24, 33, 34, 35, 36, 37, 38). The most ubiquitous is chicken GnRH II, which was first isolated from chicken brain (46). Because the chicken GnRH II structure is totally conserved from bony fish to man, this is probably the earliest evolved form and has critical functions (see Section II.C). This form has been designated GnRH II, whereas the hypothalamic form is designated type I (18, 47). In many vertebrate species, a third conserved form of GnRH (salmon GnRH) is localized to the terminal nerve in the forebrain in teleost fish and is designated GnRH III (48). GnRH III exhibits complete sequence conservation but occurs only in teleosts, suggesting that the gene encoding this peptide originated after the divergence of teleosts from the vertebrate lineage. Interestingly, sockeye salmon possess two genes encoding GnRH III. Structural analysis of the genes encoding the GnRHs supports this general classification into three forms (48), and this conclusion is confirmed by a more extensive phylogenetic analysis (Fig. 2).
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). The type 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 in tandem within single genes. The most ancient of the GnRHs identified is a homolog identified in the octopus (49). This molecule exhibits the characteristic pGlu and Pro9Gly10.NH2 but has an additional two amino acids inserted in the middle region of the molecule. Nevertheless, it is capable of stimulating LH release from quail pituitary cells (49).
B. Structure of GnRH and peptide analogs
The conservation of the length of the peptide (10 amino acids) and the NH2 terminus (pGlu-His-Trp-Ser) and COOH terminus (Pro-Gly.NH2) (Fig. 1) indicates that these features are critically important for receptor binding and activation. This is borne out from structure-activity data from several thousand analogs that were developed largely on an empirical basis. Indeed, cognizance of the evolutionary constraints on GnRH structure identify the functionally important residues and would have obviated a considerable degree of the endeavor to produce agonists and antagonists. The considerable variation in position 8 of natural GnRHs (Arg, Gln, Trp, Ser, Thr, Asn, Leu, Tyr, Lys, Ala, Trp) suggests that virtually any residue is tolerated in this position. However, this is clearly not the case for the mammalian pituitary type I GnRH receptor (18, 50), which requires Arg in position 8 for high-affinity binding. Recent work on cloned nonmammalian receptors also indicates certain specificities for the amino acid in this position (35, 51, 52, 53). Thus, the residue in position 8 seems to play an important role in ligand-selectivity of the different GnRH receptors. The roles of the individual amino acids comprising GnRH and the structure-activity relations of agonist and antagonist analogs have been extensively reviewed (18, 50) and will not be repeated here.
Short peptides such as GnRH are highly flexible in solution and exist as an equilibrium between numerous conformations (54, 55, 56, 57). However, among these conformations are so-called bioactive conformations that represent preferred structures for interaction with the receptor. For GnRH, the bioactive conformation is the product of a number of influences, which include intramolecular interactions, local influences of solvents (water), lipids, and initial receptor interactions that conform the ligand. In addition, membrane and intracellular proteins associate with the receptor and alter its conformation and selectivity for ligand conformations. Studies on GnRH and its receptor are increasingly pointing to a multiplicity of bioactive conformations of both the ligand and receptor. These in turn result in differential activation of intracellular signaling pathways (our unpublished observations).
The first studies on GnRH structure by conformational energy analysis of the NH2-terminal 1–6 and carboxyl-terminal 6–10 amino acids identified a low energy CC conformer that featured a ß-II’ type turn involving Tyr5-Gly6-Leu7-Arg8 such that the NH2 and COOH termini are closely apposed (55) (Fig. 3). This conclusion has subsequently been supported by a variety of experimental data with synthetic GnRH analogs (58, 59, 60, 61), interactions with region-specific antibodies (62), and a range of physicochemical studies. These studies have been extensively reviewed (18, 50) and will only be briefly covered here. A recent study using electron capture dissociation mass spectrometry has confirmed the presence of the ß-II’ type turn and interaction of the NH2 and COOH termini (N. C. Polfer, personal communication).
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). Yet these extended forms (e.g., Gln8GnRH) have high activity in many nonmammalian GnRH receptors (18, 51, 52, 65, 66, 67) despite their low activity at the mammalian receptor (18). The ß-II’ type turn conformation of GnRH also appears to be induced in part by the interaction of Arg8 with an acidic residue [Asp7.32(302)] in extracellular loop (EC) 3 of the mammalian receptor (18, 68, 69, 70) (Fig. 5). Substitution of a D-amino acid for Gly6 apparently enhances the ß-II’ type turn conformation and increases the activity of Arg8 GnRH about one to two orders of magnitude at mammalian receptors (18, 50). The D-amino acid substitution overcomes the deleterious effects of Arg8 substitution (e.g., with Gln8) such that binding affinity for the mammalian receptor is increased almost 1000-fold (52, 68, 69) (see Section V.A).
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The amino-terminal residues of GnRH are involved in receptor activation, and modification of these residues in GnRH produces analogs with antagonistic properties (18, 50) (Figs. 3 and 5). As in agonists, substitution of Gly6 with a D-amino acid enhances the activity of the antagonists. Because the antagonists have high binding affinity, the loss of amino-terminal contacts in agonists that activate the receptor is presumably compensated for by new contacts made by the substituted amino acids in antagonists.
The first generation of potent GnRH antagonists were characterized by high histamine-releasing properties as a result of the presence of basic residues (basic-X-basic sequence). Elimination of basicity produced analogs with lower histaminic properties but poorer solubilities and the tendency to form gels. This has resulted in difficulties in formulation that continue to be a problem in GnRH antagonists that are currently under clinical investigation. The primary structures of GnRH analogs that are extensively employed therapeutically, or are in clinical development, are shown in Fig. 5.
GnRH II is an intriguing exception to the general conclusion that a D-amino acid substitution for Gly6 enhances the binding affinity of GnRHs (72) (Table 2). An explanation may be that GnRH II is already stabilized in the ß-II’ turn conformation and that incorporation of a D-amino acid in position 6 does not further stabilize this conformation. Residues His5, Trp7, and Tyr8 are proposed to contribute to the stabilization of GnRH II (72). Gly6 is essential to allow assumption of the folded conformation, and the NH2- and COOH-terminal sequences are essential for receptor binding and activation. Thus, all the amino acids appear to have crucial roles, and this offers an explanation for the total conservation of GnRH II structure over 500 million yr of evolution.
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). The presence of chiral amino acids in place of the achiral Gly prevents the ß-II’ turn, which results in low binding affinity at the mammalian pituitary receptor (18, 50). This suggests that the receptors in these lower organisms do not have a requirement for a folded conformation of GnRH and that this feature first evolved in the receptors of the bony fish. We have recently found that replacement of Ala6 in Ciona I (Fig. 1) with Gly restores binding affinity at the human type I GnRH receptor and that substitution with D-Ala further enhances binding affinity (R. P. Millar, unpublished observations).
C. The evolutionarily conserved GnRH II
As mentioned earlier, a second form of GnRH identified from chicken brain (chicken GnRH II, GnRH II) (Fig. 1) is ubiquitous in vertebrates from primitive bony fish to man (22, 23, 24, 33, 34, 35, 36, 37, 38, 73, 74). This complete conservation of structure over 500 million yr suggests that GnRH II has an important function and a discriminating receptor (or receptors) that has selected against any structural change in the ligand. This points to essential functions that have yet to be definitively identified. The wide distribution of GnRH II in the central and peripheral nervous systems suggests a neurotransmitter/neuromodulatory role. This has been thoroughly demonstrated in the inhibition of M currents in the bullfrog sympathetic ganglion, which sensitizes neurons to depolarization (75, 76). GnRH II was identified in amphibian sympathetic ganglia, and the receptors present are highly selective for the peptide (77).
Because GnRH had been shown to have direct effects on sexual arousal in rodents (78, 79, 80) and type II GnRH is localized in brain areas associated with reproductive behavior, it was suggested that this may be a role for the peptide (22, 23, 24, 33, 79, 80). GnRH II and a GnRH II analog were both found to be potent stimulators of reproductive behavior in ring doves (23, 24), song sparrows (81), and the musk shrew (82). Recently, the cognate receptor for GnRH II was cloned from the marmoset and found to be distributed in those areas of primate brain associated with reproductive behaviors (45, 83). Infusion of GnRH II into the third ventricle of female marmosets increased sexual behavior (D. Abbott, personal communication).
In addition to its apparent role as a neuromodulator in the nervous system, GnRH II and its receptor are present in reproductive tissues (45). GnRH binding sites and antiproliferative effects of GnRH analogs have also been described in reproductive tissue tumors and their cell lines (see reviews in Refs. 6 , 8 , 11 , 13 , and 45). Interestingly, GnRH and analog binding, signaling, and pharmacological effects were not characteristic of classical hypophysial type I GnRH receptors but are more similar to type II GnRH receptors (13, 83). Although this suggests that the antitumor effects in human tissues are mediated via the type II GnRH receptors (84), the human type II receptor gene is disrupted by a frame-shift and a stop codon, and a transcript that could encode a full-length receptor or the expressed receptor protein has not been identified (85, 86 86A ). It has now become apparent that the different pharmacology of GnRH analogs in affecting pituitary function and in inhibiting proliferation of tumor cell lines can both be mediated by the human type I GnRH receptor by coupling through different signaling pathways (viz. Gq for pituitary and Gi for tumor cells). This differential coupling can be accomplished through both ligand selectivity and intracellular milieu and will be discussed later in this review (see Section VIII.A).
D. Nonpeptide GnRH antagonists
The development of nonpeptide GnRH antagonists has seen intense endeavors from the pharmaceutical industry. Representative compounds are shown in Fig. 6. The first described nonpeptide GnRH antagonist (compound 1) is a fused tetracyclic benzodiazepine that blocks ovulation in rats when given at a dose of 0.5 mg/kg (87). The antifungal drug ketoconazole (Nizoral, Janssen Pharmaceutica, Beerse, Belgium) (compound 2) was found to bind and inhibit the rat pituitary GnRH receptor with an apparent IC50 of 2 µM. Addition of a number of groups to this core structure, such as dipeptides and tripeptides related to GnRH, improved affinity to approximately 500 nM (88).
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Merck has described both indole (93) (compound 6) and quinolone-based (94, 95, 96) (compound 7) small molecule antagonists, and Abbott’s description of the ketoconazole was followed 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 a further series of derivatives of compound 6 by Merck (compound 11) with IC50 in subnanomolar concentrations and excellent oral bioavailability (99) and a series of imidazol-pyrimid-5-ones from Neurocrine (compound 12) that bind the human receptor in the low nanomolar range (100, 101). Recently, a new series of small molecule antagonists have been developed by Pfizer (102).
Although there are exceptions, the majority of the small molecule GnRH antagonists conform to a simple pharmaco-phore model. This comprises a requirement for a basic protonatable nitrogen group (optionally substituted by lipophilic groups), one or two aromatic groups, and an aliphatic lipophilic group arranged in a putative ß-turn mimetic configuration (92). Some of the small molecule antagonists have progressed to clinical trial. Takeda’s thienopyrimidinedione (TAK-013) is in phase two for endometriosis and uterine fibroids, whereas their thienopyridine-one (TAK-810) is in phase one. Neurocrine’s pyrolopyrimidone (NBI-42902) is in phase one trials for a range of reproductive indications.
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III. Structure of GnRH Receptors |
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TopAbstractI. IntroductionII. Structure of GnRHs...III. Structure of GnRH...IV. Binding of GnRH...V. Binding Interactions of...VI. Receptor ActivationVII. GnRH Receptor Mutations...VIII. Structural Correlates of...IX. Conclusions and Future...References |
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T3 gonadotrope cell line (103). This sequence was confirmed (104), and it provided the basis for the cloning of GnRH pituitary receptors from the rat (105, 106, 107), human (108, 109) (Fig. 7), sheep (110, 111), cow (112), and pig (113) that share over 80% amino acid identity. Homologs of the mammalian GnRH receptors have also been cloned from a marsupial (possum) (114 114A ), catfish (65), two forms from the 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), amberjack (CAB 65407), rubber eel (AD 49750), and seasquirt (120A ). The nonmammalian receptors with greatest homology 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 as type I GnRH receptors (Figs. 8 and 9). It is not altogether clear from homology comparisons that the classification of the mammalian and nonmammalian type I together is correct, but similarities in microdomains (e.g., EC3) support this. Because the evolutionary time separating amphibians and mammals is similar to that separating amphibians and bony fish, the poor conservation of sequence of the mammalian type I GnRH receptor with the nonmammalian receptors implies a sudden acceleration in evolutionary change in the mammals. This may have been driven by the loss of the carboxyl-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 sequence) in the carboxyl-terminal tail, which potentially conveys the possibility of coupling to MAPKs (S. R. Maudsley, unpublished observations).
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). The approach also allowed the cloning of type III GnRH receptors from the bullfrog (67). The findings along with the cloning of other GnRH receptors suggest an early evolution of the three GnRH receptor subtypes in vertebrates which parallels that of the GnRH ligands (Fig. 9). As mentioned earlier (see Section II.C), we and others have been unable to identify a human type II receptor transcript lacking a frame-shift and internal stop codon. These transcripts are therefore incapable of being translated to a full-length GPCR. This apparent silencing of the type II receptor was very paradoxical given the extraordinary conservation of the cognate GnRH II ligand from bony fish to man. Stop sites or deletions in similar positions are also present in the chimpanzee, cow, and sheep (86A ), whereas a fully functional type II receptor is present in New and Old World monkeys and the pig, as well as amphibian and reptile species (45). The gene has been completely deleted in the mouse and is apparently absent in fish (45). This intriguingly sporadic inactivation or deletion of the type II receptor gene has been reviewed and concluded to arise from plasticity in the use of the GnRH receptor subtypes for signaling by the different GnRHs (45). GnRH I, GnRH II (except in mouse), and the type I receptor have been universally conserved, in contrast to the silencing of the type II receptor in a number of species. This has apparently arisen because the type I receptor is capable of binding the GnRH II with high affinity such that it can take over the role of the type II receptor, whereas the converse cannot occur due to the high ligand selectivity of the type II receptor for GnRH II. Two other explanations have been proposed for the frame-shift and the stop codon in the human type II receptor (45). First, the frame-shift and stop codon are accommodated posttranscriptionally and during translation. Numerous mechanisms of mRNA editing have been described and could potentially repair the frame-shift and stop. In this regard we have noted transcripts that splice out the stop. Alternatively, the stop codon can be translated as a selenocysteine by means of a specific tRNA and a selenocysteine insertion sequence motif (127), which is present in the 3' untranslated region of the gene encoding human type II receptor. However, we were unable to demonstrate selenocysteine incorporation (86).
Second, a partial type II receptor is elaborated and is functional. A Kozac consensus start site follows the stop, and when this partial receptor sequence is expressed in COS cells it 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 type I expression. Could it be that the function of type II transcripts is to regulate type I expression and coupling? Their coexpression in gonadotropes suggests that this is feasible. The stop codon has arisen independently in evolution in the same vicinity and no other place in unrelated species. This might suggest that there is an advantage in having the stop codon and producing partial receptor sequences. The presence of type II receptors with or without the stop codon in closely related species suggests that any advantage of the stop is only marginal.
GnRH receptor orthologs have been identified in Drosophila melanogaster (128) and Caenorhabditis elegans (P. Swanson, unpublished observations), indicating a very early evolutionary origin. However, the cognate ligand for the Drosophila GnRH receptor homolog is not a GnRH, but is an adipokinetic hormone that has a similar structure in its length and the presence of the NH2-terminal pGlu and a COOH-terminal amide (129).
We have examined the suggested classifications of GnRH receptors by constructing phylogenetic trees. The primary amino acid structures of cloned GnRH receptors were aligned and used to generate an unrooted tree using a topological algorithm that optimizes tree structure before determining branch lengths. This revealed that the receptors can be grouped into distinct classes: types I, II, and III (Fig. 9). Type I and type II GnRH receptors form elongated clusters. Type III GnRH receptors are more closely related to type II receptors than to type I receptors, suggesting that type II and type III receptors may have arisen from duplication of an ancestral gene in lower vertebrates. The numerical naming of individual cloned receptors in the Genbank database frequently does not comply with the phylogenetic relatedness because researchers have named them by pharmacological characteristics, order of discovery, or tissue expression (67, 115, 116, 122). For example, Bogerd et al. (122) recently named a novel catfish receptor R2, although it has greatest homology with goldfish 1a receptor (51). Similarly, Wang et al. (67) named the receptor cloned from bullfrog pituitary as bullfrog I, although its greatest homology is with type 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 by seven -helical TM domains connected by three EC domains and three intracellular loop (IC) domains. The extracellular domains and superficial regions of the TMs are usually involved in binding of peptide hormones such as GnRH, and the TMs are believed to be involved in receptor configuration and conformational change associated with signal propagation (receptor activation). These changes are thought to propagate into conformational changes in the intracellular domains involved in interacting with G proteins and other proteins for intracellular signal transduction.
A unique feature of the mammalian type I GnRH receptor is the absence of a carboxyl-terminal tail present in all other GPCRs and in all of the nonmammalian and mammalian type II GnRH receptors. This is, therefore, a recently evolved feature that presumably serves an important role in the functioning of the mammalian GnRH receptor (see Section VI.F).
The conservation of amino acids during evolution from bony fish to mammals is likely to identify those residues that are crucial for GnRH receptor function (18, 35). These include residues thought to be involved in GnRH receptor binding Asp2.61(98), Asn2.65(102), Lys3.32(121), Asn5.39(212), Tyr6.58(290), and Asp7.32(302) (Figs. 7 and 8). The conserved residues include those conserved or conservatively substituted throughout the rhodopsin family of GPCRs. These are shown in Fig. 7 in squares and are the residues used as reference points for the consensus 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 I GnRH receptor
A knowledge of the three-dimensional structure of the mammalian GnRH receptor is essential for an understanding of its molecular functioning. The only direct structural information at atomic resolution of a GPCR is derived from x-ray analysis of the ground state of rhodopsin (130). Previous structural information on GPCRs was predicted from low resolution electron microscopy of bacteriorhodopsin and rhodopsin (131, 132, 133). Structural information for all other GPCRs has relied on molecular models (18, 19, 134, 135, 136, 137, 138) based on the rhodopsin structure. A model (139) incorporating structural information derived from the analyses of approximately 500 sequences in the rhodopsin-like family of GPCRs ultimately turned out to be very similar to the structure of rhodopsin, suggesting a similar structure of the seven TM domains of all GPCRs in the rhodopsin family. In contrast, the EC and IC domains are highly variable in amino acid sequence and probably in their tertiary structure. In addition to this limitation, no direct structural information of the activated state of any GPCR is available. Consequently, an understanding of the conformational changes associated with receptor activation has relied on biophysical and biochemical studies.
The development of the first published GnRH receptor molecular model was based on initial alignment and positioning of the TM helices as indicated in the projection map of the electron density of rhodopsin followed by refinement of the angles, kinking, and side chain orientation of the TMs based on the specific amino acids comprising the GnRH receptor TMs (18). The validity of the model and proposed interactions of the TM side chains was tested by site-directed mutagenesis. An example is the observation that two residues that are highly conserved in GPCRs, Asp2.50 in TM2 and Asn7.50 in TM7, appear to have undergone reciprocal mutation 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 the two residues interact with each other. Mutation of Asn2.50(87) in TM2 to Asp abolished receptor function, but a second mutation in TM7, recreating the arrangement found in other GPCRs [Asp2.50(87) and Asn7.49(318)], restored ligand binding (140). Cook et al. (141) reported that the reciprocal mutant was totally inactive, but the original observation of good binding activity (140) has been confirmed (142, 143, 144). This restoration of ligand binding by reciprocal mutation demonstrates that the side chains of two residues in TMs 2 and 7 have complementary roles in maintaining the structure of the receptor and occupy the same microenvironment within the receptor helical bundle. This experimentally derived conclusion was subsequently supported in the structural analysis of inactive rhodopsin, which shows that these residues are capable of interacting through a water molecule (145).
Recently, the rhodopsin x-ray structure has been used as a template for homology modeling of the TM domains of the GnRH receptor (146) (Z.-L. Lu, unpublished observations). Evidence gleaned from the mutagenesis of the reciprocal TM2/TM7 mutations (mentioned above) and the disulfide bridges [Cys(14)/Cys5.27(200), Cys3.25(114)/Cys5.23(196), see below] was also used. A 2.5-nsec molecular dynamic simulation of the GnRH receptor in a water-vacuum-water box with no conformational restraints during the last 2 nsec was also undertaken. This revealed a hydrogen bond net of Glu2.53(90)-Lys3.32(121)-Asp2.61(98) between TM2 and TM3 that may represent a component of intramolecular interactions that stabilize a receptor conformation similar to that of rhodopsin in the inactive state (146).
The seven TM helical domains are known from physical structural studies in the rhodopsins to be arranged in a tight bundle enclosing a hydrophilic pocket and surrounded by the hydrophobic membrane environment (18, 19, 25, 130, 133, 134, 135, 136, 137) (see schematic in Fig. 10). The evolutionary conservation of residues along a distinct face of the TM domains in the various GnRH receptors is evident (compare Figs. 7 and 8). This suggests that the conserved, more hydrophilic faces are orientated toward the hydrophilic pocket or the boundaries formed by the seven TM domains and potentially assists in the refinement of the molecular model. This proposal is supported by the studies on the TM2/TM7 interaction of Asn2.50(87) and Asp7.49(318) (140, 147) because Asn2.50(87) is clearly part of the conserved hydrophilic face of TM2.
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, 8, and 10). This was determined by a combination of photoaffinity labeling with a photoactive GnRH analog, followed by protease digestion, reduction of S-S bonds and separation of the receptor fragments by gel electrophoresis (148). The study also indicated that Cys(14) in the NH2-terminal domain and Cys5.27(200) in EC2 form a second disulfide bridge, thus further defining the position of NH2 terminus and EC2 loop structures. The highly conserved Trp4.50(164), located in the middle of TM4, may make hydrogen or Van der Waals contacts with His2.45(82) (TM2) and Met3.42(131) (TM3) because the equivalent residues in rhodopsin form a H-bond network (130). Alanine mutation of these residues in the GnRH receptor leads to a complete loss of ligand binding (Z.-L. Lu, unpublished observations), supporting the presence of this intramolecular contact as a crucial interaction for positioning of TM2 and TM4, receptor folding, and stabilization of the ground state. This TM2/TM4 interaction may be extended to TM3 via Van der Waals contact between His(82) (TM2) and Met(131) (TM3), sequestering TM3 within the bundle core and creating a buttress against whose face the other TM helices, particularly TM6 and 7, can articulate and move (149). These intramolecular contacts may also account for TM3 having the greatest tilt. The intracellular end of TM3 points into the center of the triangle formed by TMs 4, 5, and 6 of the receptor in the ground state (130). In addition, seven TM receptors such as rhodopsin may occur as dimers in native disc membranes (150). We find that TM4 of the GnRH receptor and a number of other seven TM receptors contains the G/SxxxG/S motif, which is thought to favor TM helix-helix association (151, 152), suggesting that TM4 may form a seven TM receptor homo- or heterodimer interface. Cysteine cross-linking between TM4s of the dopamine D2 receptors also suggested that the extracellular end of TM4 may form a symmetrical homodimer interface (153).
Although progress has been made in establishing molecular models of the TM helix bundle of the GnRH receptor, the proposed structure of the EC and IC is conjectural. Considerable effort has been directed at establishing programs to define loop structures (e.g., based on sequences for loop structures established from x-ray crystallography). However, these are not applicable to large loop sequences. Moreover, the known structures of the rhodopsin loops may be quite different from those of other GPCRs. Thus, a future challenge is the determination of the structure of the loops in the GnRH receptors. Some progress has been made in circular dichroism, NMR, and Raman spectral analysis of the structure of a synthetic peptide of EC3 anchored by cross-links similar to the distance between the anchoring TM6 and TM7 domains (69). Contrary to the suggestion that EC3 had an -helical structure (154), these techniques revealed the predominantly random structure of the loop. The NMR analysis showed a low incidence of a ß-hairpin structure (Fig. 11). When this structure is incorporated in the receptor model, Asp7.32(302) is able to interact with Arg8 of GnRH when docked to the other interacting sites [Asp2.61(98), Asn2.65(102), and Lys3.32(121)] (Fig. 12). Due to the low sequence homology of the EC and IC of the GnRH receptor with rhodopsin, Söderhall et al. (146) did not model the loops by using the rhodopsin structure alone but also by homology modeling of structural motifs found in the Protein Databank. These models of loop structures provide a point of departure for experimental testing. Despite the uncertainties of elements of the current GnRH molecular models, they are nevertheless providing key insight into putative mechanisms of ligand binding and receptor activation as a substrate for experimentation.
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IV. Binding of GnRH to the Mammalian Type I GnRH Receptor |
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TopAbstractI. IntroductionII. Structure of GnRHs...III. Structure of GnRH...IV. Binding of GnRH...V. Binding Interactions of...VI. Receptor ActivationVII. GnRH Receptor Mutations...VIII. Structural Correlates of...IX. Conclusions and Future...References |
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Although site-directed mutagenesis has been a useful experimental tool in defining ligand binding interactions of GnRH receptors and most GPCRs, it is important to distinguish direct receptor-ligand interactions from changes in receptor structure or conformation that indirectly affect ligand binding. One approach to this is to modify the ligand in parallel with receptor mutation (157). For example, if a mutation is thought to decrease binding affinity by disrupting a specific interaction with the ligand, then a ligand that lacks the interacting group should have similar affinity for wild-type and mutant receptors. In the course of the targeted mutation of almost one third of all the amino acid residues of the GnRH receptor, considerable advances have been made in identifying putative ligand contact sites in the mammalian GnRH receptor (Figs. 7, 12, and 13A, and Table 3). As is the case for other GPCRs that bind small peptides (158), amino acid residues in the ECs and exofacial parts of the TM helices of GnRH receptors are thought to participate in ligand binding interactions. Specific interactions of three residues, Asp2.61(98), Asn2.65(102), and Asp7.32(302), have been defined in detail, whereas residues Trp2.64(101), Lys3.32(121), Asn5.39(212), and Tyr6.58(290) have been shown to be important for binding of agonist ligands but not antagonists, and specific interactions have been proposed for these residues. These residues are discussed in numerical order, whereas other residues for which less experimental evidence is available are discussed at the end of this section.
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-NH group of His2 (159). Substituting Asp2.61(98) with uncharged amino acids led to an additional decrease in affinity for GnRH (compared with the Asp2.61(98)Glu mutant) that did not involve His2. It was concluded that the Asp2.61(98) side chain has one or more charge-dependent interactions that are important for high-affinity binding, but distinct from the interaction with His2. The computational model revealed an intramolecular salt bridge interaction of Asp2.61(98) in TM2 with Lys3.32(121) in TM3, which positions Lys3.32(121) to form a hydrogen bond with the backbone C=0 group of Ser4 in GnRH (159). The model also identified a second potential interaction between Asp2.61(98) and the backbone NH group of Trp3 (159). Interestingly, Lys3.32(121) had previously been proposed to interact with His2 of GnRH (160) (see Section VI.C). Thus, Asp2.61(98) appears to be involved in multiple interactions with GnRH (His2, Trp3, and Ser4) as well as an intramolecular interaction with Lys3.32(121). These conclusions have been incorporated into a recent refined GnRH receptor/ligand molecular model based on the crystal structure of rhodopsin (Fig. 13A) (146).
B. Asparagine2.65(102) [N2.65(102)]
An investigation of the glycosylation of the GnRH receptor showed that the Asn2.65(102) residue, located near the extracellular end of TM2, is not glycosylated, but enhanced potency of GnRH at the Asn2.65(102)Gln mutant suggested a role in ligand binding (155). Mutation of Asn2.65(102) to Ala resulted in a 27- to 750-fold loss of potency in stimulating phosphatidylinositol hydrolysis by GnRH and analogs containing the naturally occurring carboxyl-terminal Gly10-NH2 (NH-CH2-CO-NH2). The mutation had a lesser effect on the potency of analogs in which Gly10-NH2 was substituted with an ethylamide (-NH-CH2-CH3), and it was concluded that Asn2.65(102) forms a hydrogen bond with Gly10-NH2 (161), probably via the C=O group (18). Although this conclusion is reasonable, the energy attributed to the loss of a hydrogen bond is insufficient to account for the 27- to 750-fold loss in potency, and Asn2.65(102) may also be contributing to the configuration of the binding pocket. A subsequent study confirmed these findings and also showed that the binding of an antagonist, which has D-Ala10-NH2 substituted for Gly10-NH2, was decreased 2.8-fold by the Asn2.65(102)Ala mutation, consistent with possible disruption of a hydrogen bond 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 the biogenic amine receptors interacts with the positively charged amine head group of biogenic amine ligands (157). We considered that the equivalent residue [Lys3.32(121)] of the GnRH receptor may interact with GnRH. Mutation of Lys3.32(121) to Arg had minor effects on ligand binding and agonist-stimulated inositol phosphate production, whereas mutation to Asp, Ala, or Leu led to a total loss of agonist binding and inositol phosphate production. Mutation to Gln resulted in a 2000-fold reduction in agonist potency, without affecting affinity for a peptide antagonist (160). Because GnRH has no negatively charged functional groups and because peptide antagonists differ from agonists in their three amino-terminal residues, Lys3.32(121) was proposed to interact with His2 of GnRH by a charge-strengthened hydrogen bond (160). This proposal needs to be confirmed by systematic ligand modification. The subsequent demonstration that His2 of GnRH interacts with Asp2.61(98), computational modeling that showed an interaction of Lys3.32(121) with Asp2.61(98) and the similar phenotypes of mutants with uncharged substitutions for Asp2.61(98) and Lys3.32(121), led to the suggestion that Lys3.32(121) may have a role in maintaining the conformation of the agonist binding pocket and constraining the peptide backbone of the receptor (159). If this is the case, then it appears that GnRH peptide antagonist binding is not affected by these structural changes. Other computational modeling and mutagenesis studies suggested that Lys3.32(121) also interacts with Glu2.53(90) of the receptor and pGlu1 of GnRH (162). However, in more refined models, these authors suggested that His2 interacts with both Asp2.61(98) and Lys3.32(121) (146). At this stage, it is not clear whether Lys3.32(121) interacts with pGu1 or His2 or whether it has any direct interaction with agonist ligands. The very large decrease in agonist potency at the Lys3.32(121)Gln mutant (2000-fold) suggests disruption of the conformation of the ligand binding pocket or multiple interactions.
D. Asparagine5.39(212) [N5.39(212)]
Mutation of Asn5.39(212) to Ala markedly reduced activity of both agonists and antagonists, but mutation to Gln decreased agonist potency while having a minimal effect on antagonist interactions (162). This was interpreted as implying that Asn5.39(212) forms part of the agonist binding pocket, and computational modeling showed an interaction of the Asn5.39(212) side chain with the backbone C=O group of His2 of an agonist and no interaction of Asn5.39(212) with an antagonist (162). The model was subsequently modified and showed an interaction of Asn5.39(212) with pGlu1 (146, 163). The latter proposal is similar to a previously reported model of 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 substituted GnRH analogs and Asn5.39(212) mutants are required to define the role of Asn5.39(212) in ligand recognition. It should be noted that the decreased activity of both agonists and antagonists at the Asn5.39(212)Ala mutant suggests an additional role for the Asn212 side chain in binding antagonists or in receptor structure.
E. Tyrosine6.58(290) [Y6.58(290)]
Mutation of aromatic amino acids at the extracellular end of 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 and the Phe6.60(292) mutant was fully active. The Trp6.57(289) and Tyr6.58(290) mutants had reduced expression, and both mutants retained wild-type antagonist binding affinity. The Trp6.57(289) mutant showed a decrease in agonist potency (
10-fold) in a signaling assay, whereas the Tyr6.58(290) mutant showed markedly decreased potency (200- to 1000-fold) of agonists (163). This effect of mutation of Tyr6.58(290) to Ala has been independently verified (J. S. Davidson, J. Hapgood, and R. P. Millar, unpublished observations). On the basis of docking the ligand to the receptor model, it was proposed that Tyr5 of GnRH interacts with Tyr6.58(290) (163). Experimental evidence with Tyr5-substituted GnRH analogs is required to test this proposal.
F. Aspartate7.32(302) [D7.32(302)]
As described above, mammalian type I GnRH receptors preferentially 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). To investigate the possibility that Arg8 forms an electrostatic interaction with an acidic residue in the receptor, conserved acidic residues of the mouse GnRH receptor were mutated to the isosteric amide residues, Asn or Gln, and a mutant that did not discriminate Arg8-containing GnRH from uncharged [Gln8]GnRH was sought. One mutant, Glu7.32(301)Gln, had decreased affinity for mammalian GnRH, but did not change affinity for [Gln8]GnRH and increased affinity for the negatively charged [Glu8]GnRH, showing a loss of selectivity for Arg8 and gain of function for [Glu8]GnRH (68). Similarly, mutation of the equivalent residue of the human GnRH receptor [Asp7.32(302)] to Asn showed that the acidic Asp7.32(302) residue confers selectivity of the human receptor for Arg8 (70). However, although Arg8 and the acidic residue in EC3 are required for high-affinity binding of GnRH, conformationally constrained GnRH analogs (see Section II) bound the receptor with high affinity that was independent of Arg8 and/or the acidic residue (68, 70). This result indicates that once the ligand is in the high-affinity conformation, the putative interaction of Arg8 with the acidic residue does not contribute to the binding energy of the final ligand-receptor complex and suggests that the acidic residue induces or selects a ß-II’ conformation of the ligand. This observation led to the proposal that Arg8 of GnRH interacts transiently with the acidic residue to induce a high-affinity conformation of the ligand that allows it to interact with a final binding pocket, which does not include the acidic residue (70). These results suggest that caution should be exercised in using the Arg8 interaction with Asp7.32(302) as a fixed point in computational models of ligand-receptor complexes.
Although nonmammalian type I GnRH receptors do not preferentially bind Arg8-containing mammalian GnRH, the acidic residue in EC3 is conserved in these receptors (Fig. 8). In mammalian receptors, the acidic residue is followed by a Pro residue, whereas Pro precedes the acidic residue in nonmammalian receptors. The unique structural characteristics of Pro residues suggested that the position of Pro7.33(303) may be important for the selective binding of mammalian GnRH by mammalian receptors. Substituting Pro7.33(303) of the human 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 structure prediction showed that modifying Pro7.33(303) influences the conformation of EC3 (165 165A ). These results show that Pro7.33(303) may stabilize a conformation of EC3 of mammalian GnRH receptors that orients the acidic side chain to allow selective binding of mammalian GnRH.
G. Effects of mutations of other residues on the ligand binding pocket
Other receptor residues, for which less experimental evidence is available, have been proposed to be involved in ligand binding. Mutation of Trp2.64(101) to Ala resulted in a pronounced shift in agonist-induced signal transduction (162). This residue is adjacent to Asn2.65(102), which is thought to interact with Gly10NH2 of GnRH (161). The molecular model suggests that Trp2.64(101) makes a hydrogen bond with the oxygen of the Leu7 backbone of a GnRH agonist (162). However, the loss of energy associated with loss of a single hydrogen bond in the mutant cannot account for the shift in agonist-induced signal transduction of three orders of magnitude, so it is likely that mutation of Trp2.64(101) has a major effect on the formation of the binding pocket for agonists and not antagonists. Phe5.43(216) mutation to Ala had little effect on receptor function, but mutation to Tyr decreased activity about 10-fold (162). It is likely that Phe5.43(216) does not play an important role in ligand binding, but insertion of a phenolic residue disrupts receptor function.
Three computational models have proposed that Trp6.48(280) [Trp6.48(279) in the rat] in TM6 interacts with Trp (3) of GnRH (146, 164, 166). The Trp6.48 residue is also proposed to interact with a Val residue in EC3 (164) or a conserved Phe residue in TM7 (166). Mutation of Trp6.48 to Ser or Arg decreased ligand binding and abolished inositol phosphate production. The Trp6.48 residue is highly conserved among rhodopsin-like GPCRs, and the loss in signal transduction is consistent with a conserved role in receptor activation rather than specific ligand interactions.
Many mutations have been found to have no affect on ligand binding (Table 3). These residues are most probably filler residues that play little or no role in receptor function. In contrast, many other mutations (Table 3) resulted in complete loss of receptor function. These findings are not instructive because the loss of function, which may be due to destruction of the overall receptor architecture and/or binding pocket, failure to target to the plasma membrane, or instability of the receptor at the cell surface and rapid targeting to the proteosome, makes it difficult to measure parameters of ligand binding. The use of small molecule antagonists to rescue these mutants (167) may assist in determining their binding and signaling characteristics.
H. Ligand docking to the receptor
GnRH and GnRH agonists have been satisfactorily docked to receptor models via the identified binding sites (18, 159, 162, 163, 164). In the most recent GnRH receptor model, D-Trp6 GnRH was docked in the ß-II’ turned conformation to the identified contact sites in a putative active conformation of the receptor in which all of the experimentally identified binding interactions are accommodated (Fig. 13A) (146). In this model the Glu2.53(90)-Lys3.32(121)-Asp2.61(90) hydrogen bond net, identified in a previous model (163), is disturbed by His2 hydrogen bonds with Lys3.32(121) and Asp2.61(98), which are proposed to be involved in receptor activation (see Section VI). In addition to the interacting sites for the natural GnRH agonist, D-Trp6 of the superactive agonist was proposed to interact with Trp6.57(289), adjacent to Tyr6.58(290), which is proposed to interact with Tyr5 (146). The proposed interaction of the D-Trp6 side chain with Trp6.57(289) is not consistent with experimental results in which mutation of Trp6.57(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 the simulation, the C atoms of the receptor were restricted using harmonic restraints of 1 kcal mol–1 Å–2 that allow only small conformational changes of the receptor model (163), and therefore the model, in general, represents the docking of GnRH analogs to the inactive state of the receptor. However, agonist peptide binding to its receptor would lead to conformational changes of both ligand and receptor.
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V. Binding Interactions of Other GnRH Ligands and Other Receptors |
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TopAbstractI. IntroductionII. Structure of GnRHs...III. Structure of GnRH...IV. Binding of GnRH...V. Binding Interactions of...VI. Receptor ActivationVII. GnRH Receptor Mutations...VIII. Structural Correlates of...IX. Conclusions and Future...References |
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B. Peptide agonists
As mentioned above, two of the most common modifications incorporated into synthetic peptide agonists are substitution of Gly6 with a D-amino acid and Gly10.NH2 with ethylamide. Both of these modifications affect receptor interactions that have been defined for GnRH. Mutagenesis and ligand modification studies have indicated that GnRH agonists with D-amino acid substitutions for Gly6 and with no substitution of Gly10.NH2 are dependent on Asp2.61(98), Asn2.65(102), and Lys3.32(121) for binding and receptor activation (159, 160, 161, 163). Because Asp7.32(302) plays a role in interacting with Arg8 for configuring the native ligand at the receptor, this residue is not required in agonists that are configured by the D-amino acid substitution of Gly6 (68, 70). These experimentally derived conclusions contrast with molecular models in which Arg8 is modeled to interact with Asp7.32(302) for the D-Trp6 GnRH agonist (146, 162) (Fig. 13A). GnRH agonists with ethylamide substitutions for Gly10.NH2 show smaller decreases in potency at the Asn2.65(102)Ala mutant (161, 162) and probably do not interact with Asn2.65(102), whereas GnRH agonists that retain Gly10.NH2, retain normal interaction with Asn2.65(102) (Fig. 13A).
C. Peptide antagonists
Identification of binding sites for GnRH peptide antagonists has been less comprehensive than for agonists. Like GnRH, peptide antagonists of the GnRH receptor are decapeptides, but with 50–70% of amino acids substituted (Fig. 5), and they exhibit classical competitive antagonism. This would suggest that antagonists occupy binding sites that differ from but overlap the agonist binding pocket. Recent proposals that agonist and antagonist or inverse agonist ligands bind distinct active and inactive conformations of the GPCRs (169) suggest that competitive antagonism may occur without any overlap of agonist and antagonist binding sites.
Evidence for differences between the ligand binding sites of agonists and antagonists was provided by early biochemical studies. Pretreatment of pituitary membranes with proteolytic enzymes decreased binding of labeled antagonist more than that of labeled agonist. This indicated that the agonist binding site is less accessible and more buried within the receptor molecule than the antagonist binding site (170). Differences in the binding sites are also suggested by the greater effects of monovalent and divalent cations on agonist binding (170, 171). In another study, tryptic digestion of GnRH receptors that had been photoaffinity labeled with agonist or antagonist yielded different size fragments, suggesting distinct sites of attachment for agonist and antagonist ligands (172) or distinct configurations of the agonist- and antagonist-bound receptor. In this study, the photoactive agent was attached to D-Lys in position 6 of the ligands. In contrast, identical labeled receptor fragments were obtained for labeling with both a photoactive agonist (148) and a photoactive antagonist (173) when the receptor was digested with GluC to target specific cleavage sites at Glu residues. Despite the photoactive group being at position 1 of the antagonist and position 6 of the agonist, both peptides were cross-linked to Cys14 in the receptor. The identification of the same receptor residue in cross-linking two peptides with photoactive groups at different positions is consistent with the peptides having distinct binding configurations. This finding is also consistent with the concept that agonists, inverse agonists, and antagonists bind to different receptor conformations representing the continuum of equilibria between states of the active and inactive receptor. However, it may also suggest that there is considerable movement of the ligand and that covalent attachment is to the most photoreactive amino acid in the general environment (173). Thus, this kind of photoaffinity labeling may not identify precise ligand interaction sites.
The first mutation displaying differential effects on agonist and antagonist interactions was the marked effect of mutation of Lys3.32(121) to Gln on agonist potency and its failure to affect antagonist affinity. This indicated that, although Lys3.32(121) may be important for agonist binding, it clearly is not an antagonist contact site (160). Other mutations that have been shown to have differential effects on agonist and 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 mutation disrupted interaction with His2 of GnRH, a residue that is substituted with bulky D-amino acids in antagonists, so its minimal disruption of Cetrorelix binding (159) was to be expected. The Asp2.61(98) side chain is unlikely to interact with antagonists. The Trp2.64(101)Ala mutation decreased agonist potency by three orders of magnitude, but also decreased antagonist binding affinity 23-fold (162). This effect on antagonist binding may result from disruption of a direct interaction of Trp2.64(101) with the antagonist, but disruption of receptor configuration is more likely. Mutating Asn5.39(212) to Gln had minimal effects on antagonist interactions, but the Ala mutation decreased antagonist affinity 86-fold (162). This large change suggests that hydrophilic interactions at the Asn5.39(212) locus stabilize the configuration of the antagonist binding site. The Asp7.32(302)Asn mutation showed little or no effect on binding of antagonists, including two that have Arg in position 8 (70) (K. D. Pfleger, and R. P. Millar, unpublished observations). This is consistent with the proposed role of Asp7.32(302) in inducing a high-affinity ligand conformation (70). Because peptide antagonists are constrained in the high-affinity conformation, they are unlikely to interact directly with Asp7.32(302).
Docking of peptide antagonists to a GnRH receptor molecular model suggested that antagonists with Arg in position 8 [Cetrorelix (Fig. 5)] and a dicyclic peptide (174) interact with Asp7.32(302) as for Arg8 agonists (146). These researchers 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 experimentally. These researchers also propose that the NH2 terminal CO moiety of Cetrorelix interacts with Asn5.39(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 is thought to also interact with Asn5.39(212), whereas D-Cpa3 interacts with Trp6.48(280). Although the catfish GnRH receptor has low affinity for GnRH antagonist 135–18 (see Section IV.D), substitution of EC 1, 2, and 3 of the catfish GnRH receptor into the human receptor had little effect on the affinity of antagonist binding (K. D. Pfleger, and R. P. Millar, unpublished observations). This suggests that this GnRH antagonist either binds to TM residues or interacts with the few EC loop residues that are conserved between catfish and human receptors (Fig. 8).
D. Nonpeptide antagonists
The endeavor to develop orally active nonpeptide antagonists for the treatment of hormone-dependent diseases and for new generation contraceptives (14) has resulted in the development of molecules with nanomolar binding affinities (Fig. 6). Although generally less comprehensive studies have been conducted on identifying their binding sites, some indications have arisen from ligand docking to molecular models and limited mutagenesis studies.
A quinolone-based antagonist (compound 7, Fig. 6) bound the dog GnRH receptor with a 160-fold decreased affinity compared with the human receptor (175). Construction of dog/human chimeric receptors followed by site-directed mutagenesis revealed that Phe7.43(313) in TM7 of the human receptor (Leu in the dog) is responsible for the difference in affinity. Peptide agonist and antagonist analog binding was unaffected by mutation of this residue. Docking the quinolone antagonist to the human and dog receptor models showed that the quinolone ring faces Phe7.43(313) and Leu7.43(313), respectively, and the difference in surface area of these two side chains is 90 Å, which contributes 2.25 kcal/mol and accounts for the difference in binding affinity. The binding model also predicted that GnRH binding sites Lys3.32(121) and Asp7.32(302) interact with the compound and that its binding site overlaps that of GnRH. However, no direct experimental evidence (e.g., with mutant receptors) has been presented for the interaction of the quinolone antagonist with these GnRH binding sites.
A thienopyridine-based antagonist (compound 5, Fig. 6) with nanomolar affinity for the human receptor was also docked to a human GnRH receptor model (92). This proposes a hydrophobic lining to the bottom part of the binding pocket that interacts with difluorobenzyl and the thienopyridine moieties and an interaction of the positively charged amino groups with Asp7.32(302) (Fig. 6). Again, no mutagenesis studies were reported to support the proposed interactions. However, this laboratory demonstrated that the mutant Asp7.32(302)Asn exhibited a 5-fold reduction in binding affinity for the compound (R. P. Millar, unpublished observations).
Overall, the current knowledge on binding of nonpeptide small molecule GnRH antagonists indicates that the binding pocket partially overlaps that of GnRH and may also use residues crucial for peptide binding. Unlike the GnRH peptide, which predictably interacts with the same residues in mammalian GnRH receptors, some of the nonpeptide antagonists show marked species specificity (binding affinity differences of several orders of magnitude) despite sequence identity exceeding 80%. Thus, small microdomain differences such as Phe313 in the human receptor can result in large species differences in binding affinities of nonpeptide antagonists, as has been noted in a number of neuropeptide GPCRs.
E. Binding sites in nonmammalian type I GnRH receptors
The conservation of all the well-established and putative ligand 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 human type I receptor suggests that these residues serve the same function (see equivalent residues in Fig. 8). However, their functional significance has only been partially investigated and only in the chicken and catfish receptors (53) (K. D. Pfleger, unpublished observations). Although an acidic residue is present in the equivalent position of the human Asp7.32(302) in the nonmammalian type I receptors, this acidic residue would not be expected to interact with Arg8 of mammalian GnRH because these receptors do not bind mammalian GnRH with higher affinity than the other vertebrate GnRHs as occurs for the mammalian type I receptor (51, 66, 67, 71, 119, 176, 177). It was observed that a Pro residue precedes the acidic residue in EC3 of the nonmammalian type I GnRH receptors, whereas the Pro residue follows the acidic residue in mammalian type I receptors. This suggests that the orientation of the side chain of the acidic residue may effect selectivity for Arg8 GnRH. The recent demonstration that exchange of the Pro residue to the nonmammalian position in the human receptor resulted in a loss of selectivity for mammalian GnRH in the human receptor suggests that a Pro residue following the acidic residue is necessary for selective binding of mammalian GnRH by mammalian receptors (165). It has been reported that mutation of the acidic residue of the catfish GnRH receptor decreased affinity for mammalian GnRH and a series of Arg8-containing analogs, including one with a D-amino acid in position 6 (53). Because the catfish receptor has very low affinity for mammalian GnRH, this result suggests that even if 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 binding sites of the nonmammalian GnRH receptor may be configured differently from the nonmammalian receptor ligand binding sites. This is reflected in the 100-fold higher binding affinity of the catfish, chicken, and Xenopus receptors for GnRH II compared with the human GnRH receptor, despite the apparent use of the same binding sites.
F. Binding sites in type II receptors
With the exception of Asp7.32(302), all of the proposed GnRH binding sites for type I receptors are present in type II receptors (Fig. 8). These conserved sites are likely, therefore, to be involved in binding of the cognate ligand, GnRH II. The absence of Asp7.32(302) is expected because GnRH II lacks Arg8 and mutation of the acidic residue in the mouse and human type I receptors did not affect the binding of GnRH II (68, 70). The reduced binding affinity of mammalian GnRH at the marmoset (83), bullfrog (67), and Xenopus (B. Troskie, unpublished observations) type II receptors is probably partly due to the absence of Asp7.32(302), which is important for interaction with Arg8 of GnRH and high-affinity binding. Recent mutations of the marmoset type II receptor residues equivalent to Asp2.61(98), Asn2.64(102), and Lys3.32(121) show that the functions of these amino acids are similar in type I and II GnRH receptors (C. A. Flanagan, unpublished observations). The high-affinity binding of GnRH II to type II receptors and all other GnRH receptors may be due to its stabilization in the ß-II’ turn conformation due to intramolecular interactions (see Section V.A.).
G. Utilization of binding sites common to the rhodopsin family of GPCRs
There appears to be significant conservation of the binding sites for GnRH and those of other rhodopsin family GPCRs, albeit with alterations in the precise positioning and nature of the interacting residues (Table 4). Asp2.61(98) of the GnRH receptor is equivalent to functionally important residues Gln2.61(108) in the closely related vasopressin V1a receptor, and Val2.61(81) and Phe2.61(91) of the D2- and D4-dopamine receptors. Trp2.64(101) of the GnRH receptor has critical equivalents Phe2.64(86), His2.64(93), and Thr2.64(134) in the 1-adrenergic, ß2-adrenergic, and 5-hydroxytryptamine (2A) (5HT2A) receptors, respectively. Lys3.32(121) in the GnRH receptor is found in the homologous position to the Asp3.32 residue, which acts as the counter-ion of the amine group of small biogenic amines in the adrenergic-, muscarinic-, acetylcholine-, histamine-, dopamine-, and 5-hydroxytryptamine (2A) (5HT2A) receptors. In the oxytocin and vasopressin receptors, this position is occupied by Gln [Gln3.32(119), Gln3.32(131)], which is also important for ligand binding (178). Asn5.39(212) is represented by important Thr residues in position 5.39 in the muscarinic receptors and Val or Ala in the adrenergic receptors. Trp6.48(280) is conserved and shown to be crucial for function of rhodopsin, muscarinic, dopamine, serotonin, TRH, and angiotensin receptors. The equivalent residue to Asp7.32(302) in EC3 of the human GnRH receptor is conserved with the AT1-angiotensin II receptor. In both the AT1-angiotensin II- and GnRH receptors, this residue at the end of 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 the deeper binding pocket in a similar way to GnRH (179). EC3 therefore appears to have become an important interaction for small peptides (3–10 amino acids) as an adjunct to the interactions with TM residues similar to those used by biogenic amines.
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VI. Receptor Activation |
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from Gß
subunits results in GTP hydrolysis, reformation of the G-GDPß complex, and a return of the receptor to the inactive (R) state (181). The Samama-revised model proposes that receptors fluctuate between an inactive R conformation and an active R* conformation (169). The R* conformation has high affinity for agonists and productively stimulates G protein turnover. By stabilizing the R* conformation, agonists shift the equilibrium further to R* conformers. The models are essentially the same in that they both require conformational change in the receptor, one ligand-induced (conformation induction) and the other ligand-stabilized (conformation selection). Kenakin (182) proposed that GPCRs may occur in multiple conformations, and the equilibria are controlled by the interactions of strategically key residues (183). Agonist binding 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 signal selectivity or LISS, because both agonists and classical antagonists can selectively signal. For example, we have shown that certain antagonists for the Gq pathway (inositol phosphate production) at the type I GnRH receptor can act as agonists on the Gi pathway as measured by their capacity to inhibit forskolin-mediated cAMP accumulation in various reproductive tissue backgrounds (Ref. 185 , and L. Davidson, manuscript in preparation). Similarly, with respect to ligand-specific signaling events, we have also found that GnRH I activates Src whereas GnRH II is inhibitory on Src at the type I GnRH receptor (S. R. Maudsley, unpublished observations). The mechanism of agonist-induced receptor activation is thought to result from a disruption of the intramolecular constraint networks that stabilize the ground state of the receptor. The disruption of subsequent replacement by a new set of contacts results in the stabilization of the active conformation of the receptor allowing binding of signal-mediating proteins to intracellular domains of the receptor. Valuable information about conformational change associated with receptor activation emerged from the pioneering site-directed double spin labeling studies of rhodopsin (186). These indicated that receptor activation involves rotations of the TM helices and outward movements of the endofacial parts of TMs 2, 3, 6, and 7. These helices are packed tightly in the ground state but open up in the activated state. The predominant movement of the TM helices is an approximate 30° clockwise rotation (viewed from the cytoplasmic surface) of the endofacial part of TM6 relative to TM3 (187). This accomplishes an 8 Å outward movement of TM6 toward the intracellular end of TM5 (186). The closure of the intracellular ends of TM5 and 6 during receptor activation was further confirmed by agonist-induced double-cysteine cross-linking in the M3 muscarinic acetylcholine (188). A small outward movement (about 2–4 Å) of TM2 toward TM4, and TM7 toward TM6 also occurs during photoactivation of rhodopsin (186). There is probably a small movement and rotation of TM3, which accompanies the movement 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 rearranged into the middle of TM2 and TM4. This brings TM3 more perpendicular to the plane of the membrane and closer to TM7, thereby eliminating cavities at the intracellular end of TM3. Evidence of the movement of TM3 also emerged in a recent cross-linking experiment in which a photoreactive retinal analog labeled TM6 in the dark state as the crystal structure predicts, but reacted with TM4 after light activation (192). In the crystal structure, a modeled all-trans retinal conformation passes through TM3, and therefore TM3 must be nudged away from this area to open a way between TM7 and TM4 (191). The reconfiguring of TM3 is also consistent with the presence of cavities at the intracellular end of TM3.
A. Interaction of Asn2.50(87)/Asp7.49(319) in TM2/7 in GnRH receptor activation
Mutation of Asn2.50(87) and Asp7.49(318) in the mouse GnRH receptor [Asn2.50(87) and Asp7.49(319) in the human] (Figs. 7 and 8) revealed that Asp7.49(318) is involved in receptor activation (signal propagation) because the mutants Asn2.50(87)Asn7.49(318) and Asp2.50(87)Asn7.49(318) both retained good ligand binding but poor stimulation of inositol phosphate production (140). These findings indicate that the unusual arrangement of having Asp7.49 in TM7 in the GnRH receptor is an essential component of ligand-mediated receptor activation, which normally is subserved by the conserved Asp2.50 in TM2 of other GPCRs and the nonmammalian GnRH receptors (121) (Fig. 8).
Interestingly, the presence of an Asp7.49 in TM7 of the GnRH receptor facilitates coupling to phospholipase C (PLC) via Gq/11 but prevents coupling to phospholipase D (PLD) by the small monomeric G protein (147). Mutation to Asn7.49, which is present in the majority of GPCRs, recreates this coupling to PLD. Thus, the reverse arrangement of Asn2.50 and Asp7.49 in TM2 and TM7 in the GnRH receptor appears to have been selected to allow PLC coupling and prevent PLD coupling (147). Further exploration by mutation of TM2 Asn2.50 and TM7 Asp7.49 to various amino acids has confirmed that the TM2 Asn2.50 is essential for configuring and expression of the receptor, whereas the TM7 Asp7.49 is only essential for receptor activation (143). Thus, these early studies revealed that Asn2.50 in TM2 and Asp7.49 in TM7 are an element of the molecular switch of receptor activation.
B. Disruption of TM3 Asp3.49(138)/Arg3.50(139) interaction in GnRH receptor activation
The highly conserved motif DRxxxI/V at the intracellular end 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 a charge interaction (193), and this has been confirmed in the crystal structure of rhodopsin (130). Disruption of this by mutating Asp to an uncharged residue in the GnRH receptor conveys increased coupling efficiency, possibly through the release of Arg3.50(139) to interact with other residues (193). This suggests that the Asp3.49(138)Arg350(139) charge interaction is disrupted in the active state of the receptor. The Ile3.54(143) located one helical turn below the Arg350(139) appears to play a role in caging the Arg3.50(139) side chain for coupling interactions by sterically limiting its movement. Mutation to small residues (e.g., Ala) results in some uncoupling (193). It is proposed that the Arg3.50(139) side chain is involved in a triad interaction with the TM2 Asn2.50 and TM7 Asp7.49 in stabilizing the active conformation of the receptor (193) (Fig. 14B). The Arg3.50(139) is crucial for coupling because mutation to Gln leads to very poor coupling efficiency (193). The receptor activation-induced movements of TM helices described above may lead to the Arg3.50(139) side chain interacting with Tyr7.53(323) in the conserved TM7 N/DPxxY motif in the active conformation, which was proposed by Vriend and colleagues (195). This highly conserved Tyr7.53 is critically important for G protein signaling in the GnRH receptor (144) and in other rhodopsin family GPCRs (196, 197, 198, 199, 200, 201).
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interactions, which may account for the distinctive results caused by mutagenesis of both residues. Thus, an essential element of activation of the GnRH receptor and other GPCRs appears to be an agonist-induced (or stabilized) disruption of intramolecular constraint networks accompanied by the protonation of Asp3.49(138) to release Arg3.50(139) for interaction with the Asn2.50(87)/Asp7.49(319) residues in TM2 and TM7 and possibly Tyr7.53(323). Because the conserved Asn1.50 in TM1 of rhodopsin interacts with Asp2.50 in TM2 in the inactive state (130), this residue may also play a role in the TM domain network involved in receptor activation. We have been unable to explore this because mutation of Asn1.50(53) leads to an absence of detectable binding and signaling, which may be due to nonfunctional receptor or poor binding.
C. The triad of Glu2.53(90)-Lys3.32(121)-Asp2.61(98)
Flanagan et al. (159) have produced experimental evidence suggesting an interaction of Asp2.61(98) with Lys3.32(121). Studies on a GnRH receptor model have proposed that an H-bond network of Glu2.53(90)-Lys3.32(121)-Asp2.61(98) is present in the inactive state (Figs. 13B and 14A) and is replaced by a new set 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 molecular switch in agonist activation of the receptor (146). This hypothesis is supported by the demonstration that Lys3.32(121) is essential for agonist binding but not antagonist binding (160) and the observation of a 100-fold decrease in affinity of native GnRH (His2) binding by the Asp2.61(98)Glu mutant and maintenance of mutant binding affinity for Trp2 GnRH (159). Glu2.53(90) mutation to Gln had no effect on function (68), but mutation to Ala resulted in complete loss of function (162). Presumably Gln2.53(90) is able to maintain hydrogen bonding with Lys3.32(121).
D. Role of extracellular loop 2
The disulfide bridge between the extracellular end of TM3 and EC2 is conserved in all of the GPCRs in the rhodopsin family and is essential for receptor function (202). Because Lys3.32(121) and Asp3.49(138)/Arg3.50(139) in TM3 play a major role in receptor activation, the rigid connection of TM3 to EC2 through the disulfide bridge suggests an important role of this extracellular domain in the assumption of the active and inactive states of the receptor. This supposition is supported by reports that antibodies against EC2 of the 1 (203), ß1- and ß2-adrenergic (204), AT1-angiotensin II (205), bradykinin B2 (206), and M1- (207) and M2-muscarinic-acetylcholine (208) receptors can activate second messenger responses, presumably by stabilizing the receptor in the active conformation. Changes in the EC2 configuration might translate into stabilizing TM3, TM4, and TM5 in the active conformation due to its physical connection to these domains. The crystal structure of rhodopsin reveals that EC2 has a distinct structure intimately associated with the TM domains.
Strong evidence has been obtained for a role of EC2 in the activation of the human GnRH receptor (209). Certain antagonists at the human GnRH receptor are agonists at the chicken (71) and Xenopus (209) receptors. Similarly, incorporation of chicken receptor EC domains into the human receptor established that this domain is responsible for conferring agonist activity to a GnRH antagonist (71). By incorporating the Xenopus GnRH receptor extracellular domains into the human GnRH receptor, Ott et al. (209) demonstrated that the Xenopus 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 of Val5.26(197)Ala together with Trp5.32(205)His was sufficient to convey agonism to antagonist 135–18. By comparing agonistic behavior of a range of GnRH antagonists, a single residue D-Lys(iPr) in position 6 was shown to be responsible for the phenomenon (209). Together with the pH dependence of the effect, the findings suggested that His5.32(205) forms a charge-supported hydrogen bond with D-Lys(iPr)6 of the antagonist to stabilize the receptor in the active conformation. The recent demonstration that a single mutation of Ala5.25(201) to Thr in EC2 in combination with a TM6 mutation in the frog GnRH receptor alters signaling further underlines the importance of EC2 in receptor activation (115).
E. Other residues possibly involved in receptor activation
Other potential elements of GnRH receptor activation may include the interactions between Asn5.39(212) of TM5 and pGlu1 of GnRH, and between Tyr6.58(290) and Tyr5 of GnRH (162, 163). In another GnRH receptor model, Trp (3) of GnRH was predicted to penetrate about 20 Å into the TM core and interact with Trp6.48(279) [Trp6.48(280) in the human receptor] of TM6 (164) and potentially intercalate between the indole moiety of Trp6.48(279) and the phenyl moiety of Phe7.41(310) [Phe7.41(311) in the human receptor] in TM7 (166). Mutation of Trp6.48(279) to Ser or Phe7.41(310) to Leu (present in most GPCRs) reduced binding affinity slightly but totally abrogated coupling to inositol phosphate production. Because mutation of both residues together was not additive, it was interpreted that they interact and constitute part of the activation network. Trp6.48 in other GPCRs (e.g., rhodopsin, cholecystokinin B, and angiotensin AT1A receptors) plays a crucial role in receptor function (210, 211, 212).
F. Integrated model of GnRH receptor activation
The data described above point to a number of intramolecular changes associated with the interconversion of active and inactive configurations of the human GnRH receptor. Precisely how these changes relate to each other in receptor activation is currently conjectural. We propose that in the inactive state Asp3.49(138) forms a charge interaction with Arg3.50(139), Glu2.53(90)-Lys3.32(121)-Asp2.61(98) form a hydrogen bond network, and Asn2.50(87) interacts with Asn1.50(53) and Asp7.49(319) (Fig. 15). In the active configuration, Asp3.49(138) is protonated, releasing Arg3.50(139) to interact with Asn2.50(87), Asp7.49(319), and possibly Tyr7.53(323) (Fig. 15). Asn2.50(87) may also interact with Asn1.50(53) in TM1 in the active conformation because this residue is crucial for receptor function. These new interactions may be accompanied by a loss of interaction of the Glu2.53(90)-Lys3.32(121)-Asp2.61(98) triad, allowing the binding of GnRH and the formation of the contact Lys3.32(121)-His2(GnRH)-Asp2.61(98). These changes give rise to a rotation of TM3, which affects the other TMs directly (e.g., TM4 and TM5 through the disulfide bridge between TM3 and EC2) and indirectly through interhelical interactions [e.g., TM2 and TM7 through Asn2.50(87) and Asp7.49(319) interactions]. These helical movements are thought to be associated with a different configuration of the connected ICs, which favors binding/activation of intracellular signaling proteins.
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), which is present in a number of GPCRs. Like the majority of GPCRs, and contrary to the mammalian GnRH receptor, the Asp2.50 in TM2, and not the Asp7.49 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 mammalian type I GnRH receptors in which the Asp7.49 in TM7 is essential for coupling. Interestingly, the total loss of function of the catfish receptor TM2 Asp2.50(90)Asn mutant can be partially restored by mutation of a Met2.53(93) one helical turn above to Glu as in the mammalian GnRH receptor [Glu2.53(90), Fig. 8] (168). It appears therefore that the unusual reciprocal arrangement of these interacting TM2/TM7 residues in the mammalian receptor is related to the loss of the carboxyl-terminal tail. We propose that the efficient functioning of the mammalian type I GnRH receptor required the loss of the carboxyl-terminal tail such that it did not undergo rapid desensitization (see Section VIII.C). The loss of the tail became the driving force for coordinated molecular changes to the receptor. |
VII. GnRH Receptor Mutations in Hypogonadotropic Hypogonadism |
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TopAbstractI. IntroductionII. Structure of GnRHs...III. Structure of GnRH...IV. Binding of GnRH...V. Binding Interactions of...VI. Receptor ActivationVII. GnRH Receptor Mutations...VIII. Structural Correlates of...IX. Conclusions and Future...References |
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). These patients are characterized by delayed sexual development and inappropriately low or apulsatile gonadotropin and sex steroid hormone levels in the absence of functional abnormalities of the hypothalamopituitary axis (223). Although some of the mutants are totally nonfunctional 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 have some ability to elicit an inositol phosphate response to GnRH [Asn(10)Lys, Thr(32)Ile, Gln2.69(106)Arg, Arg6.30(262)Gln, and Tyr6.52(284)Cys] (see Table 3 for references).
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Recently, a cell permeant small molecule antagonist (compound 11 in Fig. 6) was shown to rescue all of the naturally occurring mutants except Ser4.54(168)Arg, Ser5.44(217)Arg, and Leu7.44(314)Stop by increasing their expression (167). These findings suggest that the majority of mutations result in an instability or misfolding of the GnRH receptor and that the small molecule antagonist stabilizes these mutants and protects them from targeting to degradative pathways in the endoplasmic reticulum (224). These intriguing observations offer the possibility of using cell-permeant small molecule antagonists as therapeutics for GnRH receptor and other GPCR mutations, and also as a valuable tool for characterizing the properties of poorly expressed experimental mutants.
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VIII. Structural Correlates of GnRH Receptor Coupling and Internalization |
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TopAbstractI. IntroductionII. Structure of GnRHs...III. Structure of GnRH...IV. Binding of GnRH...V. Binding Interactions of...VI. Receptor ActivationVII. GnRH Receptor Mutations...VIII. Structural Correlates of...IX. Conclusions and Future...References |
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, resulting in the hydrolysis of membrane-bound phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-triphosphate and diacylglycerol, which mobilize intracellular calcium and activate PKC, respectively. These in turn stimulate the biosynthesis and secretion of the gonadotropins, LH and FSH. These intracellular signaling pathways have been reviewed extensively (27, 29, 30, 225, 226, 227 227A ) and will not be discussed further here. Instead, in this section we focus our attention on the structural features of GnRH receptors that determine their coupling to different intracellular proteins mediating intracellular signaling and internalization of the receptors. Additionally, we consider the new concept that different GnRH ligands can determine preferential interactions with different intracellular proteins through stabilization of the GnRH receptor in different conformations. Our recent unpublished observations have led us to formulate a novel concept which proposes that the GnRH receptor can assume a number of different active conformations that preferentially and selectively activate different intracellular 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 active conformations are stabilized (activated) by different GnRH analogs. The challenge now will be to determine the nature of these specific ligand-receptor interactions and receptor conformations that transduce specific signalling (which we have termed GnRH ligand-selective signalling) and physiological and pathophysiological effects.
A. Coupling to multiple G proteins
Gq/11 is the predominant G protein coupled to the GnRH receptor in various cellular environments (27, 225, 228). A number of studies have demonstrated that other G proteins can mediate the actions of GnRH receptors. Pretreatment of rat pituitary cells with pertussis toxin decreased inositol phosphate production in response to GnRH, suggesting coupling to either Gi or Go (229). In addition, GnRH receptor coupling to Gi has been demonstrated in ovarian carcinomas (230, 231, 232), uterine leiomyosarcomas (230), uterine endometrial carcinomas (232, 233), and human prostate cancer cells (234). Pretreatment of rat pituitary cells with cholera toxin results in an increase in GnRH stimulation of LH, suggesting coupling to Gs (235, 236). In addition, Gs and Gi coupling had been revealed by the GnRH stimulation of cAMP in a number of experimental paradigms (226, 237, 238, 239, 240, 241). What are the structural features of the GnRH receptor that facilitate these differences in coupling?
The ICs and carboxyl-terminal tail have been implicated in specific coupling of GPCRs to G proteins, but their degree of involvement varies among different receptors. Because all the mammalian GnRH receptors lack a carboxyl-terminal tail, effective receptor-G protein interactions must take place via one or more of the ICs. The conservation of the carboxyl-terminal sequence of IC3 in vertebrate GnRH receptors (Fig. 8) suggested that this region may be crucial for coupling to the primary mediator, Gq/11. A series of cassette substitutions covering the entire sequence of IC3 confirmed this hypothesis (I. Wakefield, unpublished observations). Within this region, Ala6.29(261) was identified as an important residue for coupling. When the equivalent Ala in IC3 of biogenic amine receptors is mutated to large residues, the receptors are constitutively active (242). However, mutation of Ala6.29(261) to bulky amino acids resulted in an opposite effect in the GnRH receptor, namely uncoupling of the receptor and failure to generate inositol phosphate (243). Mutation of the evolutionarily conserved adjacent basic amino acid [Arg6.30(262)] to Ala was also shown to result in uncoupling (I. Wakefield, unpublished observations), and natural mutations of Arg6.30(262) have been shown to cause uncoupling in receptors of families with hypogonadotrophic hypogonadism (213, 216, 244). The effects of overexpression of rat GnRH receptor IC3 peptides in GnRH receptor-expressing GGH3 cells on inositol phosphate production and cAMP accumulation demonstrated that this domain is involved in coupling to Gq/11 and Gs signal transduction pathways (245).
The GnRH receptors have the conserved motif DRxxxI/VxxPL at the N terminus of IC2, which plays a role in coupling. The importance of the DRxxxI element in receptor activation (see Section VI) appears to extend to the Pro3.57(146)Leu3.58(147). Mutation of the preceding Arg3.56(145) to Pro causes uncoupling (246), presumably because this mutation introduces a Pro-Pro motif known to disrupt secondary structure. Replacement of the conserved Leu3.58(147) with Asp or Ala led to defective Gq/11 coupling (247), and mutation of Arg3.50(139) of the mouse GnRH receptor to Gln produced a similar effect (194).
In IC1, the sequence (KKLSR) is a Gs recognition motif (BBxxB, where B is a basic amino acid); mutation of certain of these residues leads to uncoupling of cAMP production but not of inositol phosphate production (239).
The above studies indicate that the GnRH receptor is able to couple to several G proteins and activate a number of effectors via different elements of the three ICs. It appears that coupling to Gq/11 occurs through IC2 and IC3, and to Gs through IC1. Coupling to Gi is less understood, but may be related to the cell-type, stage of the cell cycle, or availability of the Gi protein. Recently, it has been suggested that Gi activation underlies the antiproliferative effects of GnRH in many cancers (231, 248). The IC elements for Gi coupling have not been investigated.
The carboxyl-terminal tail of GPCRs has also been implicated in the regulation of signaling via receptor-coupled G proteins. In contrast to the mammalian GnRH receptors, but in common with other GPCRs, the cloned nonmammalian GnRH receptors all have a carboxyl-terminal tail (18, 51, 65, 67, 71). The absence of a carboxyl-terminal tail in mammalian GnRH receptors is correlated with a lack of rapid desensitization (249), in contrast to nonmammalian and type II tailed receptors that exhibit rapid desensitization. A number of studies (250, 251, 252, 253, 254) involving truncation, site-directed mutagenesis, and carboxyl-terminal tail-swapping have established the importance of this region for coupling, desensitization (reviewed in Ref. 255), and receptor internalization (see Section VIII.C). Although mammalian receptors lack the carboxyl tail, the carboxyl-terminal residues of TM7 are important for Gq/11 effector coupling (256). Because the last four residues (YFSL) of all mammalian GnRH receptors are a conserved putative class II postsynaptic density, discs-large, ZO-1 (PDZ) domain-binding motif, these residues may be important for effective mammalian GnRH receptor signal transduction.
B. Regulators of G protein signaling (RGS) proteins
Inactive G proteins are heterotrimers consisting of the -, ß-, and -subunits. Upon receptor activation, GTP displaces GDP from binding to the G-subunit and is then hydrolyzed to GDP by intrinsic GTPase activity. This promotes the reassociation of G-subunit and ß-dimers, forming the inactive heterotrimer. RGS proteins interact directly with active G-subunits to accelerate their intrinsic GTPase activity and limit their half-life (257). Two family members, RGS3 and RGS10, have been implicated in the regulation of GnRH receptor coupling (258, 259, 260). Furthermore, there is evidence that the carboxyl-terminal tails of nonmammalian GnRH receptors may be sites for interactions with RGS10, although the nature of this interaction is unclear (260).
In addition to RGS proteins, there is scope and precedence for involvement of accessory proteins such as arrestins, GPCR kinases, Src-homology domain 2 domain-containing proteins, small GTP-binding proteins (261), polyproline-binding proteins, receptor-activity-modifying proteins, and members of the scaffolding family of proteins such as PDZ domain-containing proteins (262) in the regulation GnRH receptor coupling and signal transduction. The requisite sequence structural motifs within the GnRH receptors have not been identified.
C. GnRH receptor internalization
At least four pathways of agonist-induced internalization of GPCRs exist (263), which may be cell-type specific. The classical GPCR internalization pathway involves GPCR kinases, ß-arrestin, clathrin-coated pits, and the GTPase dynamin, and is exemplified by the ß2-adrenergic receptor (263, 264, 265, 266, 267, 268). Upon receptor activation, G protein-coupled receptor kinases are targeted to the receptor by the generation of free ß-subunits. Activated G protein-coupled receptor kinases phosphorylate the receptor at specific serine and threonine residues. Receptor phosphorylation enhances the binding of ß-arrestin. ß-Arrestin binding interdicts G protein coupling and also serves to target the receptor to clathrin-coated pits for internalization. Dynamin is thought to be necessary for the scission of clathrin-coated vesicles from the plasma membrane (269). GPCRs have also been reported to internalize independently of both ß-arrestin and dynamin, or in pathways dependent on only one or the other, implying that GPCRs are able to undergo internalization via pathways that are distinct from clathrin-coated pits (263, 264, 265, 266, 267). The subcellular localization of certain GPCRs to smooth noncoated membrane structures and vesicles (270, 271) suggests that GPCRs can use internalization pathways that are distinct from clathrin-coated vesicles. Caveolae are flask-shaped, nonclathrin-coated structures that have been implicated in the internalization of small molecules and certain GPCRs (272, 273, 274, 275, 276, 277). In addition, dynamin has been implicated in caveolae function, although its exact functional role is not known. As for clathrin-coated pits, dynamin may be responsible for the pinching off of caveolae from the plasma membrane (278, 279).
The internalization pathways used by GnRH receptors are cell-type dependent and also differ between different receptor subtypes. The rat GnRH receptor, which lacks a cytoplasmic tail, internalizes in a ß-arrestin-independent manner, but probably via a clathrin-dependent mechanism (250), and in a ß-arrestin-independent pathway that is dynamin-dependent (253). The lack of a carboxyl-terminal domain in the mammalian GnRH receptors probably accounts for their ß-arrestin independency for internalization. A comparison of the pathways of internalization of the human and Xenopus type I GnRH receptors in HeLa cells reported that the human GnRH receptor internalizes in a dynamin-independent manner, whereas the Xenopus type I GnRH receptor internalizes in a dynamin-dependent manner (176). Despite the differences, both appear to internalize via a pathway that is clathrin-mediated (176). Similarly, rat GnRH receptors colocalize with transferrin receptors that are known to internalize in clathrin-coated vesicles (250). Based on the above studies, it appears that mammalian and nonmammalian GnRH receptors can both be targeted for clathrin-mediated internalization.
The carboxyl-terminal tail of the catfish GnRH receptor is important for cell surface expression, ligand binding, and receptor phosphorylation and internalization (121, 252). Agonist-induced internalization of the catfish GnRH receptor (252) is dependent on a serine residue in the carboxyl-terminal tail that is phosphorylated and may function as a ß-arrestin binding site. Addition of the carboxyl-terminal tail of the catfish GnRH receptor and TSH-releasing hormone receptor to the rat GnRH receptor results in an increased rate of internalization (280, 281).
The chicken GnRH receptor (71) exhibits rapid internalization kinetics and was shown to be dependent on the carboxyl-terminal tail for this process (251). A threonine-doublet (Thr369Thr370) located at the distal end of the cytoplasmic tail is critical, because its mutation to Ala residues abolished rapid internalization (254). The chicken GnRH receptor preferentially undergoes rapid agonist-induced internalization in a dynamin- and caveolae-dependent manner, based on the finding that internalization is inhibited in the presence of dominant negative (K44A) dynamin-1 and caveolin-1(
1–81) overexpression, and pretreatment with the caveolae disruptors, filipin and methyl-ß-cyclodextrin (254). However, internalization of the chicken GnRH receptor in COS-7 cells was independent of ß-arrestin and clathrin-coated vesicles (254).
A recent study has characterized the internalization pathways of the three bullfrog GnRH receptor subtypes (282). The bullfrog type II GnRH receptor (reclassified as bullfrog type I here; see Fig. 8) showed the most rapid rate and highest extent of internalization among the three receptors. Furthermore, internalization of the bullfrog type I GnRH receptor was shown to be both ß-arrestin and dynamin-dependent, whereas bullfrog type II and III GnRH receptors internalize via a pathway that is ß-arrestin-independent, but dynamin-dependent, similar to the pathway used by the chicken GnRH receptor (254, 282). It is interesting to note that the last eight residues of the carboxyl-terminal tails of the chicken and 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 was identified as critical for chicken GnRH receptor internalization (254). The above studies thus suggest that the carboxyl-terminal tail of the nonmammalian GnRH receptors plays a pivotal role in their function and subcellular trafficking.
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IX. Conclusions and Future Perspectives |
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TopAbstractI. IntroductionII. Structure of GnRHs...III. Structure of GnRH...IV. Binding of GnRH...V. Binding Interactions of...VI. Receptor ActivationVII. GnRH Receptor Mutations...VIII. Structural Correlates of...IX. Conclusions and Future...References |
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Acknowledgments |
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Footnotes |
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Abbreviations: EC, Extracellular loop; GPCR, G protein-coupled receptor; 5HT2A, 5-hydroxytryptamine (2A); IC, intracellular loop; NMR, nuclear magnetic resonance; PLC, phospholipase C; PLD, phospholipase D; RGS, regulator of G protein signaling; TM, transmembrane.
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References |
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TopAbstractI. IntroductionII. Structure of GnRHs...III. Structure of GnRH...IV. Binding of GnRH...V. Binding Interactions of...VI. Receptor ActivationVII. GnRH Receptor Mutations...VIII. Structural Correlates of...IX. Conclusions and Future...References |
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  N. T Joseph, K. Morgan, R. Sellar, D. McBride, R. P Millar, and I. C Dunn The chicken type III GnRH receptor homologue is predominantly expressed in the pituitary, and exhibits similar ligand selectivity to the type I receptor J. Endocrinol., July 1, 2009; 202(1): 179 - 190. [Abstract] [Full Text] [PDF]
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 S. Y. Ko, H. Guo, N. Barengo, and H. Naora Inhibition of Ovarian Cancer Growth by a Tumor-Targeting Peptide That Binds Eukaryotic Translation Initiation Factor 4E Clin. Cancer Res., July 1, 2009; 15(13): 4336 - 4347. [Abstract] [Full Text] [PDF]
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 J. Bouligand, C. Ghervan, J. A. Tello, S. Brailly-Tabard, S. Salenave, P. Chanson, M. Lombes, R. P. Millar, A. Guiochon-Mantel, and J. Young Isolated Familial Hypogonadotropic Hypogonadism and a GNRH1 Mutation N. Engl. J. Med., June 25, 2009; 360(26): 2742 - 2748. [Abstract] [Full Text] [PDF]
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 J. A. Tello and N. M. Sherwood Amphioxus: Beginning of Vertebrate and End of Invertebrate Type GnRH Receptor Lineage Endocrinology, June 1, 2009; 150(6): 2847 - 2856. [Abstract] [Full Text] [PDF]
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E. Jardon-Valadez, A. Aguilar-Rojas, G. Maya-Nunez, A. Leanos-Miranda, A. Pineiro, P M. Conn, and A. Ulloa-Aguirre Conformational effects of Lys191 in the human GnRH receptor: mutagenesis and molecular dynamics simulations studies J. Endocrinol., May 1, 2009; 201(2): 297 - 307. [Abstract] [Full Text] [PDF]
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 H.-M. Wu, H.-S. Wang, H.-Y. Huang, Y.-K. Soong, C. D MacCalman, and P. C K Leung GnRH signaling in intrauterine tissues Reproduction, May 1, 2009; 137(5): 769 - 777. [Abstract] [Full Text] [PDF]
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 S. P. Armstrong, C. J. Caunt, and C. A. McArdle Gonadotropin-Releasing Hormone and Protein Kinase C Signaling to ERK: Spatiotemporal Regulation of ERK by Docking Domains and Dual-Specificity Phosphatases Mol. Endocrinol., April 1, 2009; 23(4): 510 - 519. [Abstract] [Full Text] [PDF]
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 A. K. Roseweir, A. S. Kauffman, J. T. Smith, K. A. Guerriero, K. Morgan, J. Pielecka-Fortuna, R. Pineda, M. L. Gottsch, M. Tena-Sempere, S. M. Moenter, et al. Discovery of Potent Kisspeptin Antagonists Delineate Physiological Mechanisms of Gonadotropin Regulation J. Neurosci., March 25, 2009; 29(12): 3920 - 3929. [Abstract] [Full Text] [PDF]
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 M. Lindemans, F. Liu, T. Janssen, S. J. Husson, I. Mertens, G. Gade, and L. Schoofs Adipokinetic hormone signaling through the gonadotropin-releasing hormone receptor modulates egg-laying in Caenorhabditis elegans PNAS, February 3, 2009; 106(5): 1642 - 1647. [Abstract] [Full Text] [PDF]
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 D.-K. Kim, J. S. Yang, K. Maiti, J.-I. Hwang, K. Kim, D. Seen, Y. Ahn, C. Lee, B.-C. Kang, H. B. Kwon, et al. A Gonadotropin-Releasing Hormone-II Antagonist Induces Autophagy of Prostate Cancer Cells Cancer Res., February 1, 2009; 69(3): 923 - 931. [Abstract] [Full Text] [PDF]
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 L. F. Canosa, N. Stacey, and R. E. Peter Changes in brain mRNA levels of gonadotropin-releasing hormone, pituitary adenylate cyclase activating polypeptide, and somatostatin during ovulatory luteinizing hormone and growth hormone surges in goldfish Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2008; 295(6): R1815 - R1821. [Abstract] [Full Text] [PDF]
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 J. S. Schneider and E. F. Rissman Gonadotropin-releasing hormone II: a multi-purpose neuropeptide Integr. Comp. Biol., November 1, 2008; 48(5): 588 - 595. [Abstract] [Full Text] [PDF]
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J. A. Tello, S. Wu, J. E. Rivier, and N. M. Sherwood Four functional GnRH receptors in zebrafish: analysis of structure, signaling, synteny and phylogeny Integr. Comp. Biol., November 1, 2008; 48(5): 570 - 587. [Abstract] [Full Text] [PDF]
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C. D. White, M. Coetsee, K. Morgan, C. A. Flanagan, R. P. Millar, and Z.-L. Lu A Crucial Role for G{alpha}q/11, But Not G{alpha}i/o or G{alpha}s, in Gonadotropin-Releasing Hormone Receptor-Mediated Cell Growth Inhibition Mol. Endocrinol., November 1, 2008; 22(11): 2520 - 2530. [Abstract] [Full Text] [PDF]
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 P.-S. Tsai and L. Zhang The Emergence and Loss of Gonadotropin-Releasing Hormone in Protostomes: Orthology, Phylogeny, Structure, and Function Biol Reprod, November 1, 2008; 79(5): 798 - 805. [Abstract] [Full Text] [PDF]
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L. L. Burger, D. J. Haisenleder, K. W. Aylor, and J. C. Marshall Regulation of Intracellular Signaling Cascades by GNRH Pulse Frequency in the Rat Pituitary: Roles for CaMK II, ERK, and JNK Activation Biol Reprod, November 1, 2008; 79(5): 947 - 953. [Abstract] [Full Text] [PDF]
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R. Lopez de Maturana, A. J. Pawson, Z.-L. Lu, L. Davidson, S. Maudsley, K. Morgan, S. P. Langdon, and R. P. Millar Gonadotropin-Releasing Hormone Analog Structural Determinants of Selectivity for Inhibition of Cell Growth: Support for the Concept of Ligand-Induced Selective Signaling Mol. Endocrinol., July 1, 2008; 22(7): 1711 - 1722. [Abstract] [Full Text] [PDF]
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S. Wen, J. R. Schwarz, D. Niculescu, C. Dinu, C. K. Bauer, W. Hirdes, and U. Boehm Functional Characterization of Genetically Labeled Gonadotropes Endocrinology, June 1, 2008; 149(6): 2701 - 2711. [Abstract] [Full Text] [PDF]
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K. D. G. Pfleger, A. J. Pawson, and R. P. Millar Changes to Gonadotropin-Releasing Hormone (GnRH) Receptor Extracellular Loops Differentially Affect GnRH Analog Binding and Activation: Evidence for Distinct Ligand-Stabilized Receptor Conformations Endocrinology, June 1, 2008; 149(6): 3118 - 3129. [Abstract] [Full Text] [PDF]
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 L. H. Heitman, K. Ye, J. Oosterom, and A. P. IJzerman Amiloride Derivatives and a Nonpeptidic Antagonist Bind at Two Distinct Allosteric Sites in the Human Gonadotropin-Releasing Hormone Receptor Mol. Pharmacol., June 1, 2008; 73(6): 1808 - 1815. [Abstract] [Full Text] [PDF]
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A. J. Pawson, E. Faccenda, S. Maudsley, Z.-L. Lu, Z. Naor, and R. P. Millar Mammalian Type I Gonadotropin-Releasing Hormone Receptors Undergo Slow, Constitutive, Agonist-Independent Internalization Endocrinology, March 1, 2008; 149(3): 1415 - 1422. [Abstract] [Full Text] [PDF]
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I. Casella, H. Lindner, C. Zenzmaier, D. Riitano, P. Berger, and T. Costa Non-Gonadotropin-Releasing Hormone-Mediated Transcription and Secretion of Large Human Glycoprotein Hormone {alpha}-Subunit in Human Embryonic Kidney-293 Cells Endocrinology, March 1, 2008; 149(3): 1144 - 1154. [Abstract] [Full Text] [PDF]
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A. R Finch, K. R Sedgley, C. J Caunt, and C. A McArdle Plasma membrane expression of GnRH receptors: regulation by antagonists in breast, prostate, and gonadotrope cell lines J. Endocrinol., February 1, 2008; 196(2): 353 - 367. [Abstract] [Full Text] [PDF]
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A. J. Stewart, R. Sellar, D. J. Wilson, R. P. Millar, and Z.-L. Lu Identification of a Novel Ligand Binding Residue Arg38(1.35) in the Human Gonadotropin-Releasing Hormone Receptor Mol. Pharmacol., January 1, 2008; 73(1): 75 - 81. [Abstract] [Full Text] [PDF]
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 C. Metallinou, B. Asimakopoulos, A. Schroer, and N. Nikolettos Gonadotropin-Releasing Hormone in the Ovary Reproductive Sciences, December 1, 2007; 14(8): 737 - 749. [Abstract] [PDF]
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S. Gardner, S. Maudsley, R. P. Millar, and A. J. Pawson Nuclear Stabilization of {beta}-Catenin and Inactivation of Glycogen Synthase Kinase-3{beta} by Gonadotropin-Releasing Hormone: Targeting Wnt Signaling in the Pituitary Gonadotrope Mol. Endocrinol., December 1, 2007; 21(12): 3028 - 3038. [Abstract] [Full Text] [PDF]
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A. Caraty, J. T. Smith, D. Lomet, S. Ben Said, A. Morrissey, J. Cognie, B. Doughton, G. Baril, C. Briant, and I. J. Clarke Kisspeptin Synchronizes Preovulatory Surges in Cyclical Ewes and Causes Ovulation in Seasonally Acyclic Ewes Endocrinology, November 1, 2007; 148(11): 5258 - 5267. [Abstract] [Full Text] [PDF]
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C. A. Flanagan, C.-C. Chen, M. Coetsee, S. Mamputha, K. E. Whitlock, N. Bredenkamp, L. Grosenick, R. D. Fernald, and N. Illing Expression, Structure, Function, and Evolution of Gonadotropin-Releasing Hormone (GnRH) Receptors GnRH-R1SHS and GnRH-R2PEY in the Teleost, Astatotilapia burtoni Endocrinology, October 1, 2007; 148(10): 5060 - 5071. [Abstract] [Full Text] [PDF]
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 S Darby, J Stockley, M M Khan, C N Robson, H Y Leung, and V J Gnanapragasam Expression of GnRH type II is regulated by the androgen receptor in prostate cancer Endocr. Relat. Cancer, September 1, 2007; 14(3): 613 - 624. [Abstract] [Full Text] [PDF]
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 P. M. Conn, A. Ulloa-Aguirre, J. Ito, and J. A. Janovick G Protein-Coupled Receptor Trafficking in Health and Disease: Lessons Learned to Prepare for Therapeutic Mutant Rescue in Vivo Pharmacol. Rev., September 1, 2007; 59(3): 225 - 250. [Abstract] [Full Text] [PDF]
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T. A. Kohout, Q. Xie, S. Reijmers, K. J. Finn, Z. Guo, Y.-F. Zhu, and R. S. Struthers Trapping of a Nonpeptide Ligand by the Extracellular Domains of the Gonadotropin-Releasing Hormone Receptor Results in Insurmountable Antagonism Mol. Pharmacol., August 1, 2007; 72(2): 238 - 247. [Abstract] [Full Text] [PDF]
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 Z.-L. Lu, M. Coetsee, C. D. White, and R. P. Millar Structural Determinants for Ligand-Receptor Conformational Selection in a Peptide G Protein-coupled Receptor J. Biol. Chem., June 15, 2007; 282(24): 17921 - 17929. [Abstract] [Full Text] [PDF]
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S. Lariviere, G. Garrel, V. Simon, J.-W. Soh, J.-N. Laverriere, R. Counis, and J. Cohen-Tannoudji Gonadotropin-Releasing Hormone Couples to 3',5'-Cyclic Adenosine-5'-Monophosphate Pathway through Novel Protein Kinase C{delta} and -{epsilon} in L{beta}T2 Gonadotrope Cells Endocrinology, March 1, 2007; 148(3): 1099 - 1107. [Abstract] [Full Text] [PDF]
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 T. Ikemoto and M. K. Park Comparative analysis of the pituitary and ovarian GnRH systems in the leopard gecko: signaling crosstalk between multiple receptor subtypes in ovarian follicles J. Mol. Endocrinol., February 1, 2007; 38(2): 289 - 304. [Abstract] [Full Text] [PDF]
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Z. Naor, H. N. Jabbour, M. Naidich, A. J. Pawson, K. Morgan, S. Battersby, M. R. Millar, P. Brown, and R. P. Millar Reciprocal Cross Talk between Gonadotropin-Releasing Hormone (GnRH) and Prostaglandin Receptors Regulates GnRH Receptor Expression and Differential Gonadotropin Secretion Mol. Endocrinol., February 1, 2007; 21(2): 524 - 537. [Abstract] [Full Text] [PDF]
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R. S. Struthers, Q. Xie, S. K. Sullivan, G. J. Reinhart, T. A. Kohout, Y.-F. Zhu, C. Chen, X.-J. Liu, N. Ling, W. Yang, et al. Pharmacological Characterization of a Novel Nonpeptide Antagonist of the Human Gonadotropin-Releasing Hormone Receptor, NBI-42902 Endocrinology, February 1, 2007; 148(2): 857 - 867. [Abstract] [Full Text] [PDF]
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 E. L. Baldwin, I. N. Wegorzewska, M. Flora, and T. J. Wu Regulation of Type II Luteinizing Hormone-Releasing Hormone (LHRH-II) Gene Expression by the Processed Peptide of LHRH-I, LHRH-(1-5) in Endometrial Cells Experimental Biology and Medicine, January 1, 2007; 232(1): 146 - 155. [Abstract] [Full Text] [PDF]
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S. Mamputha, Z.-l. Lu, R. W. Roeske, R. P. Millar, A. A. Katz, and C. A. Flanagan Conserved Amino Acid Residues that Are Important for Ligand Binding in the Type I Gonadotropin-Releasing Hormone (GnRH) Receptor Are Required for High Potency of GnRH II at the Type II GnRH Receptor Mol. Endocrinol., January 1, 2007; 21(1): 281 - 292. [Abstract] [Full Text] [PDF]
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K. R Sedgley, A. R Finch, C. J Caunt, and C. A McArdle Intracellular gonadotropin-releasing hormone receptors in breast cancer and gonadotrope lineage cells J. Endocrinol., December 1, 2006; 191(3): 625 - 636. [Abstract] [Full Text] [PDF]
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 L. Lin, G. S. Conway, N. R. Hill, M. T. Dattani, P. C. Hindmarsh, and J. C. Achermann A Homozygous R262Q Mutation in the Gonadotropin-Releasing Hormone Receptor Presenting as Constitutional Delay of Growth and Puberty with Subsequent Borderline Oligospermia J. Clin. Endocrinol. Metab., December 1, 2006; 91(12): 5117 - 5121. [Abstract] [Full Text] [PDF]
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K. Morgan, R. Sellar, A. J. Pawson, Z.-L. Lu, and R. P. Millar Bovine and Ovine Gonadotropin-Releasing Hormone (GnRH)-II Ligand Precursors and Type II GnRH Receptor Genes Are Functionally Inactivated Endocrinology, November 1, 2006; 147(11): 5041 - 5051. [Abstract] [Full Text] [PDF]
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 A. Bowen, S. Khan, L. Berghman, J. D. Kirby, R .P. Wettemann, and J. A. Vizcarra Immunization of pigs against chicken gonadotropin-releasing hormone-II and lamprey gonadotropin-releasing hormone-III: Effects on gonadotropin secretion and testicular function J Anim Sci, November 1, 2006; 84(11): 2990 - 2999. [Abstract] [Full Text] [PDF]
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M. Shimizu and G. Y. Bedecarrats Identification of a Novel Pituitary-Specific Chicken Gonadotropin-Releasing Hormone Receptor and Its Splice Variants Biol Reprod, November 1, 2006; 75(5): 800 - 808. [Abstract] [Full Text] [PDF]
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B. Levavi-Sivan, J. Biran, and E. Fireman Sex Steroids Are Involved in the Regulation of Gonadotropin-Releasing Hormone and Dopamine D2 Receptors in Female Tilapia Pituitary Biol Reprod, October 1, 2006; 75(4): 642 - 650. [Abstract] [Full Text] [PDF]
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A. Granger, C. Bleux, M.-L. Kottler, S. J. Rhodes, R. Counis, and J.-N. Laverriere The LIM-Homeodomain Proteins Isl-1 and Lhx3 Act with Steroidogenic Factor 1 to Enhance Gonadotrope-Specific Activity of the Gonadotropin-Releasing Hormone Receptor Gene Promoter Mol. Endocrinol., September 1, 2006; 20(9): 2093 - 2108. [Abstract] [Full Text] [PDF]
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 A. Antelli, L. Baldazzi, A. Balsamo, P. Pirazzoli, A. Nicoletti, M. Gennari, and A. Cicognani Two novel GnRHR gene mutations in two siblings with hypogonadotropic hypogonadism. Eur. J. Endocrinol., August 1, 2006; 155(2): 201 - 205. [Abstract] [Full Text] [PDF]
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 B.C. Tarlatzis, B.C. Fauser, E.M. Kolibianakis, K. Diedrich, P. Devroey, and , On Behalf of the Brussels GnRH Antagonist Consen GnRH antagonists in ovarian stimulation for IVF Hum. Reprod. Update, July 1, 2006; 12(4): 333 - 340. [Abstract] [Full Text] [PDF]
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M R Silver and S A Sower Functional characterization and kinetic studies of an ancestral lamprey GnRH-III selective type II GnRH receptor from the sea lamprey, Petromyzon marinus. J. Mol. Endocrinol., June 1, 2006; 36(3): 601 - 610. [Abstract] [Full Text] [PDF]
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A. Leanos-Miranda, A. Ulloa-Aguirre, L. A Cervini, J. A. Janovick, J. Rivier, and P M. Conn Identification of new gonadotrophin-releasing hormone partial agonists. J. Endocrinol., June 1, 2006; 189(3): 509 - 517. [Abstract] [Full Text] [PDF]
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H. Zemkova, A. Balik, Y. Jiang, K. Kretschmannova, and S. S. Stojilkovic Roles of Purinergic P2X Receptors as Pacemaking Channels and Modulators of Calcium-Mobilizing Pathway in Pituitary Gonadotrophs Mol. Endocrinol., June 1, 2006; 20(6): 1423 - 1436. [Abstract] [Full Text] [PDF]
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J. Roa, E. Vigo, J. M. Castellano, V. M. Navarro, R. Fernandez-Fernandez, F. F. Casanueva, C. Dieguez, E. Aguilar, L. Pinilla, and M. Tena-Sempere Hypothalamic Expression of KiSS-1 System and Gonadotropin-Releasing Effects of Kisspeptin in Different Reproductive States of the Female Rat Endocrinology, June 1, 2006; 147(6): 2864 - 2878. [Abstract] [Full Text] [PDF]
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Y. L. Pagan, S. S. Srouji, Y. Jimenez, A. Emerson, S. Gill, and J. E. Hall Inverse Relationship between Luteinizing Hormone and Body Mass Index in Polycystic Ovarian Syndrome: Investigation of Hypothalamic and Pituitary Contributions J. Clin. Endocrinol. Metab., April 1, 2006; 91(4): 1309 - 1316. [Abstract] [Full Text] [PDF]
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J. A. Janovick, P. E. Knollman, S. P. Brothers, R. Ayala-Yanez, A. S. Aziz, and P. M. Conn Regulation of G Protein-coupled Receptor Trafficking by Inefficient Plasma Membrane Expression: MOLECULAR BASIS OF AN EVOLVED STRATEGY J. Biol. Chem., March 31, 2006; 281(13): 8417 - 8425. [Abstract] [Full Text] [PDF]
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K.-Y. Kim, K.-C. Choi, N. Auersperg, and P. C K Leung Mechanism of gonadotropin-releasing hormone (GnRH)-I and -II-induced cell growth inhibition in ovarian cancer cells: role of the GnRH-I receptor and protein kinase C pathway. Endocr. Relat. Cancer, March 1, 2006; 13(1): 211 - 220. [Abstract] [Full Text] [PDF]
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C. J. Caunt, A. R. Finch, K. R. Sedgley, L. Oakley, L. M. Luttrell, and C. A. McArdle Arrestin-mediated ERK Activation by Gonadotropin-releasing Hormone Receptors: RECEPTOR-SPECIFIC ACTIVATION MECHANISMS AND COMPARTMENTALIZATION J. Biol. Chem., February 3, 2006; 281(5): 2701 - 2710. [Abstract] [Full Text] [PDF]
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D. K. Barnett, T. M. Bunnell, R. P. Millar, and D. H. Abbott Gonadotropin-Releasing Hormone II Stimulates Female Sexual Behavior in Marmoset Monkeys Endocrinology, January 1, 2006; 147(1): 615 - 623. [Abstract] [Full Text] [PDF]
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M. Enomoto, M. Utsumi, and M. K. Park Gonadotropin-Releasing Hormone Induces Actin Cytoskeleton Remodeling and Affects Cell Migration in a Cell-Type-Specific Manner in TSU-Pr1 and DU145 Cells Endocrinology, January 1, 2006; 147(1): 530 - 542. [Abstract] [Full Text] [PDF]
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K. E. Ratcliffe, H. M. Fraser, R. Sellar, J. Rivier, and R. P. Millar Bifunctional Gonadotropin-Releasing Hormone Antagonist-Progesterone Analogs with Increased Efficacy and Duration of Action Endocrinology, January 1, 2006; 147(1): 571 - 579. [Abstract] [Full Text] [PDF]
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C. Klausen, T. Tsuchiya, J. P. Chang, and H. R. Habibi PKC and ERK are differentially involved in gonadotropin-releasing hormone-induced growth hormone gene expression in the goldfish pituitary Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2005; 289(6): R1625 - R1633. [Abstract] [Full Text] [PDF]
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P. E. Barran, R. W. Roeske, A. J. Pawson, R. Sellar, M. T. Bowers, K. Morgan, Z.-L. Lu, M. Tsuda, T. Kusakabe, and R. P. Millar Evolution of Constrained Gonadotropin-releasing Hormone Ligand Conformation and Receptor Selectivity J. Biol. Chem., November 18, 2005; 280(46): 38569 - 38575. [Abstract] [Full Text] [PDF]
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N. J. Westphal and A. F. Seasholtz Gonadotropin-Releasing Hormone (GnRH) Positively Regulates Corticotropin-Releasing Hormone-Binding Protein Expression via Multiple Intracellular Signaling Pathways and a Multipartite GnRH Response Element in {alpha}T3-1 Cells Mol. Endocrinol., November 1, 2005; 19(11): 2780 - 2797. [Abstract] [Full Text] [PDF]
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 N. Wettschureck and S. Offermanns Mammalian G Proteins and Their Cell Type Specific Functions Physiol Rev, October 1, 2005; 85(4): 1159 - 1204. [Abstract] [Full Text] [PDF]
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J. A. Tello, J. E. Rivier, and N. M. Sherwood Tunicate Gonadotropin-Releasing Hormone (GnRH) Peptides Selectively Activate Ciona intestinalis GnRH Receptors and the Green Monkey Type II GnRH Receptor Endocrinology, September 1, 2005; 146(9): 4061 - 4073. [Abstract] [Full Text] [PDF]
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Z.-L. Lu, R. Gallagher, R. Sellar, M. Coetsee, and R. P. Millar Mutations Remote from the Human Gonadotropin-releasing Hormone (GnRH) Receptor-binding Sites Specifically Increase Binding Affinity for GnRH II but Not GnRH I: EVIDENCE FOR LIGAND-SELECTIVE, RECEPTOR-ACTIVE CONFORMATIONS J. Biol. Chem., August 19, 2005; 280(33): 29796 - 29803. [Abstract] [Full Text] [PDF]
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S. Shacham, M. N. Cheifetz, M. Fridkin, A. J. Pawson, R. P. Millar, and Z. Naor Identification of Ser153 in ICL2 of the Gonadotropin-releasing Hormone (GnRH) Receptor as a Phosphorylation-independent Site for Inhibition of Gq Coupling J. Biol. Chem., August 12, 2005; 280(32): 28981 - 28988. [Abstract] [Full Text] [PDF]
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M. R. Silver, N. V. Nucci, A. R. Root, K. L. Reed, and S. A. Sower Cloning and Characterization of a Functional Type II Gonadotropin-Releasing Hormone Receptor with a Lengthy Carboxy-Terminal Tail from an Ancestral Vertebrate, the Sea Lamprey Endocrinology, August 1, 2005; 146(8): 3351 - 3361. [Abstract] [Full Text] [PDF]
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P. E. Knollman, J. A. Janovick, S. P. Brothers, and P. M. Conn Parallel Regulation of Membrane Trafficking and Dominant-negative Effects by Misrouted Gonadotropin-releasing Hormone Receptor Mutants J. Biol. Chem., July 1, 2005; 280(26): 24506 - 24514. [Abstract] [Full Text] [PDF]
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N. Moncaut, G. Somoza, D. M Power, and A. V M Canario Five gonadotrophin-releasing hormone receptors in a teleost fish: isolation, tissue distribution and phylogenetic relationships J. Mol. Endocrinol., June 1, 2005; 34(3): 767 - 779. [Abstract] [Full Text] [PDF]
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A. J. Pawson, S. Maudsley, K. Morgan, L. Davidson, Z. Naor, and R. P. Millar Inhibition of Human Type I Gonadotropin-Releasing Hormone Receptor (GnRHR) Function by Expression of a Human Type II GnRHR Gene Fragment Endocrinology, June 1, 2005; 146(6): 2639 - 2649. [Abstract] [Full Text] [PDF]
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M. M. Grumbach A Window of Opportunity: The Diagnosis of Gonadotropin Deficiency in the Male Infant J. Clin. Endocrinol. Metab., May 1, 2005; 90(5): 3122 - 3127. [Abstract] [Full Text] [PDF]
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A. Leanos-Miranda, A. Ulloa-Aguirre, J. A. Janovick, and P. M. Conn In Vitro Coexpression and Pharmacological Rescue of Mutant Gonadotropin-Releasing Hormone Receptors Causing Hypogonadotropic Hypogonadism in Humans Expressing Compound Heterozygous Alleles J. Clin. Endocrinol. Metab., May 1, 2005; 90(5): 3001 - 3008. [Abstract] [Full Text] [PDF]
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V. M. Navarro, J. M. Castellano, R. Fernandez-Fernandez, S. Tovar, J. Roa, A. Mayen, M. L. Barreiro, F. F. Casanueva, E. Aguilar, C. Dieguez, et al. Effects of KiSS-1 Peptide, the Natural Ligand of GPR54, on Follicle-Stimulating Hormone Secretion in the Rat Endocrinology, April 1, 2005; 146(4): 1689 - 1697. [Abstract] [Full Text] [PDF]
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J. H. Li, H. Choe, A. F. Wang, K. Maiti, C. Wang, A. Salam, S. Y. Chun, W.-K. Lee, K. Kim, H. B. Kwon, et al. Extracellular Loop 3 (EL3) and EL3-Proximal Transmembrane Helix 7 of the Mammalian Type I and Type II Gonadotropin-Releasing Hormone (GnRH) Receptors Determine Differential Ligand Selectivity to GnRH-I and GnRH-II Mol. Pharmacol., April 1, 2005; 67(4): 1099 - 1110. [Abstract] [Full Text] [PDF]
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K.-Y. Kim, K.-C. Choi, S.-H. Park, N. Auersperg, and P. C. K. Leung Extracellular Signal-Regulated Protein Kinase, But Not c-Jun N-Terminal Kinase, Is Activated by Type II Gonadotropin-Releasing Hormone Involved in the Inhibition of Ovarian Cancer Cell Proliferation J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1670 - 1677. [Abstract] [Full Text] [PDF]
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I. S. Parhar, S. Ogawa, and Y. Sakuma Three GnRH receptor types in laser-captured single cells of the cichlid pituitary display cellular and functional heterogeneity PNAS, February 8, 2005; 102(6): 2204 - 2209. [Abstract] [Full Text] [PDF]
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S. Maudsley, L. Davidson, A. J. Pawson, R. Chan, R. L. de Maturana, and R. P. Millar Gonadotropin-Releasing Hormone (GnRH) Antagonists Promote Proapoptotic Signaling in Peripheral Reproductive Tumor Cells by Activating a G{alpha}i-Coupling State of the Type I GnRH Receptor Cancer Res., October 15, 2004; 64(20): 7533 - 7544. [Abstract] [Full Text] [PDF]
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R. P. Millar and A. J. Pawson Outside-In and Inside-Out Signaling: The New Concept that Selectivity of Ligand Binding at the Gonadotropin-Releasing Hormone Receptor Is Modulated by the Intracellular Environment Endocrinology, August 1, 2004; 145(8): 3590 - 3593. [Full Text] [PDF]
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