Conformational Constraint of Mammalian, Chicken, and Salmon GnRHs, But Not GnRH II, Enhances Binding at Mammalian and Nonmammalian Receptors: Evidence for Preconfiguration of GnRH II

Kevin D. G. Pfleger, Jan Bogerd and Robert P. Millar

Medical Research Council Human Reproductive Sciences Unit (K.D.G.P., R.P.M.), Edinburgh, EH16 4SB, United Kingdom; and Department of Endocrinology (J.B.), Utrecht University, 3508 TB, Utrecht, The Netherlands

Address all correspondence and requests for reprints to: Robert P. Millar, Director, Medical Research Council Human Reproductive Sciences Unit, Edinburgh, EH16 4SB, United Kingdom. E-mail: r.millar{at}hrsu.mrc.ac.uk.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mammalian GnRH (mGnRH) is believed to interact with mGnRH type I receptors in a ß-II' turn conformation involving residues 5–8. This conformation can be constrained by substitution of a D-amino acid at position 6 or by a lactam ring involving residues 6 and 7, thereby increasing receptor binding affinity. It has been proposed that this is not the case for non-mGnRH receptors. However, we show that this conformational constraint increases the binding affinity of mammalian, chicken, and salmon GnRH for the chicken and catfish receptors, as well as for the mouse receptor. Therefore, we conclude that the ß-II' turn conformation enhances ligand binding for non-mGnRH as well as mGnRH type I receptors. In contrast, most substitutions of a D-amino acid in position 6 have limited effect on binding affinity for GnRH II. We suggest that this ligand is preconfigured through intramolecular interactions, which accounts for its high binding affinity and total conservation of primary structure over 500 million years of evolution.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
MAMMALIAN GnRH (mGnRH) is a decapeptide released from the hypothalamus to interact with cognate receptors and regulate LH and FSH release from pituitary gonadotroph cells (1). Fifteen structural variants of GnRH have been identified (2, 3). In jawed vertebrates these cluster into three groupings: GnRH I, GnRH II, and GnRH III (4, 5). One of these, GnRH II, is totally conserved in structure from bony fish to man, suggesting that each individual amino acid is essential for biological activity (6). We propose that the N and C termini, which are conserved between all GnRHs, are involved in receptor binding and activation as for mGnRH, whereas the central residues are crucial for the configuration of GnRH II and appropriate presentation of the N and C termini.

The bioactive conformation of mGnRH acting on mGnRH type I receptors was originally proposed to be largely determined by a ß-II' turn involving residues 5–8 (7). This conclusion was supported by a large body of evidence derived from a variety of approaches. Conformation-dependent mGnRH antisera that bind the N and C termini of mGnRH tolerate certain amino acid substitutions in the central region of the ligand, but not in other positions. This implied that mGnRH has a turn conformation resulting in closely apposed N and C termini (8).

Fluorescence measurements of Trp3 at different pH values suggested that His2 and Tyr5 are in close proximity to Arg8 in mGnRH (9). This correlated with the proposed ß-II' turn conformation and indicated the possibility of intramolecular stabilizing interactions between these residues. Further fluorescence studies supported these findings by comparing mGnRH and analogs with substitutions for Arg8 (10). Chicken GnRH I (cGnRH I, [Gln8]-GnRH) was found to lack such stabilizing interactions, implying that a less structured conformation exists for this ligand.

The technique of conformational memories further supported the concept that a predominant ß-II' turn conformer of mGnRH accounts for biological activity (11). Substitution of Arg8 with Lys resulted in a loss of this structure and reduced binding affinity (11, 12).

Nuclear magnetic resonance (NMR) studies have provided direct structural evidence for mGnRH in a ß-type turn conformation consisting of three families (13). All possess the ß-type turn about Gly6 and at least two hydrogen bonds: one between Ser4 and Arg8 and another between pGlu1 and Gly10-NH2. Hydrogen bonding of Arg8 to either His2 or Tyr5 was also noted, supporting the conclusions from the fluorescence studies (9, 10). cGnRH I had different conformers grouped into four families. These also possessed several hydrogen bonds, but only that between pGlu1 and Ser4 was common to all four. This implied much greater flexibility of this molecule compared with mGnRH.

A series of cyclic GnRH antagonists were found to have a ß-II' turn conformation, further supporting the notion that this is the biologically relevant structure (14). However, because antagonists occupy different, but overlapping, binding sites, these observations do not necessarily support the structure of agonists. Indeed, in this study a potent cyclic antagonist was found to have a ß-I' turn at residues 6–7. This implies that, for antagonists at least, the specific turn type may not be important as long as it results in correct presentation of the backbone and side chains critical for binding. Because agonists must satisfactorily bind and activate the receptor, they are likely to have more specific structural requirements.

D-Amino acid substitution for Gly6 (D-aa6 substitution) is believed to stabilize the ß-II' turn conformation, thereby increasing affinity for the receptor (15). Conformational energy analysis indicates that D-aa6 substitution reduces the freedom for opening at position 6 so that the population of the bioactive conformer is increased (16). A similar effect can be achieved by utilizing a lactam ring between residues 6 and 7 (6,7 {gamma}-lactam insertion) (17).

Although there is substantial evidence for the ß-II' turn conformation for active GnRH analogs at the mGnRH type I receptor, it is uncertain whether constraint of nonmammalian GnRHs in this conformation enhances their activity at mammalian and nonmammalian receptors.

mGnRH type I and non-mGnRH receptors have low sequence identity and structural features that suggest they may be configured differently. These include differences in intracellular domains (absence of C-terminal tail, longer intracellular loop 1 in mGnRH type I receptors), extracellular disulfide bridges, and different interactions between residues in transmembrane domains 2 and 7 (18, 19). In view of these differences that affect the overall three-dimensional structure of the receptor, the bioactive ligand conformational requirement may also differ (20).

The present study provides evidence that a major determinant of bioactive conformation at mGnRH type I receptors, the ß-II' turn involving residues 5–8, is also necessary for high-affinity binding at non-mGnRH receptors. The findings also indicate that, unlike the other GnRHs, the native GnRH II ligand is preconfigured in a bioactive conformation, which may account for its relatively high affinity for all GnRH receptors investigated and the conservation of its structure over 500 million yr of evolution.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The mouse GnRH receptor binds mGnRH and GnRH II with highest affinity (Table 1Go). The catfish and chicken receptors bind GnRH II with highest affinity, followed by salmon GnRH (sGnRH), mGnRH, and cGnRH I (Table 1Go). Substitution of a D-amino acid in position 6 of the ligand (D-aa6) or the insertion of a {gamma}-lactam moiety between residues 6 and 7 (6, 7 {gamma}-lactam) significantly increases the binding affinity of mGnRH, cGnRH I, and sGnRH acting on the mouse, chicken, and catfish GnRH receptors (Table 1Go).


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Table 1. Summary of Ligand Binding to Mouse, Chicken, and Catfish GnRH Receptors

 
Introduction of the aromatic D-Trp6 residue into mGnRH increased the binding affinity at the mouse, chicken, and catfish GnRH receptors by a substantial 74-, 8.6-, and 14-fold. The affinity of [D-Trp6]-GnRH binding at the mouse receptor is significantly higher than that of [D-Ala6]-GnRH and [6,7 {gamma}-lactam]-GnRH (P < 0.05). In contrast, substitution with the positively charged D-Lys6 residue enhanced the binding affinity at these receptors by only 1.9-, 3.6-, and 2-fold.

GnRH II was unique among the natural GnRHs tested in binding all three species of GnRH receptor with relatively high affinity. The affinity for the nonmammalian receptors was particularly high. Substitution of a D-aa6 has little or no effect on the binding affinity of the GnRH II ligand acting at the catfish and chicken receptors. At the mouse receptor, which has a much lower affinity for GnRH II, only D-Lys6 substitution results in a substantial (8.4-fold) increase in affinity (Table 1Go).

The latter finding suggested that the D-Lys6 in GnRH II may be interacting with one of the seven extracellular domain acidic residues that are conserved in mGnRH type I receptors. Binding of [D-Lys6]-GnRH to mutant mouse GnRH receptors, in which each of these acidic residues was successively mutated to its isosteric amide (21), was compared with wild-type receptors. In initial binding assays the Glu8Gln, Glu111Gln, Asp185Asn, Asp292Asn, Glu294Gln, and Glu301Gln mutant receptors specifically bound radioligand, whereas the Asp98 Asn mutant receptor did not (data not shown). The latter result is expected as the Asp98 residue in the mGnRH type I receptor has been shown to interact with the His2 residue in the GnRH ligand, and mutations of this residue severely affect ligand binding (22). The mutant receptors that bound the labeled ligand were all found to have the same binding affinity as the wild-type receptor for GnRH II and [D-Lys6]-GnRH II (Table 2Go). This indicates that an interaction of D-Lys6 with one of these residues was not responsible for the increased affinity of [D-Lys6]-GnRH II.


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Table 2. Summary of Ligand Binding to Point-Mutated Mouse GnRH Receptors

 
D-Lys6 substitution substantially enhanced the binding affinity of GnRH II acting at the mGnRH type I receptor, but not at the non-mGnRH receptors. To examine this further, four more species of GnRH receptor were investigated: two mGnRH type I receptors (rat and human) and two non-mGnRH receptors (Xenopus II and bullfrog III). D-aa6 Substitution did not significantly increase the binding affinity of the GnRH II ligand acting at the Xenopus II or bullfrog III receptors (Table 3Go). D-Lys6 substitution substantially increased the binding affinity of the GnRH II ligand acting at the rat and human receptors (33.8- and 7.2-fold, respectively), as noted for the mouse receptor. Another basic amino acid (D-Arg6) substitution into GnRH II also increases binding affinity at the rat receptor.


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Table 3. Summary of Ligand Binding to Rat, Human, Xenopus II, and Bullfrog III GnRH Receptors

 
Substitution of a basic D-aa6 into GnRH II enhances binding at the rat receptor more than at the mouse or human receptors. Comparison of the amino acid sequences of the human, mouse, and rat GnRH receptor extracellular loops reveals an additional acidic residue in the rat receptor: the residues at the homologous position to Gln208 in the human are Gln207 in the mouse and Glu207 in the rat (Table 4Go). A human GnRH receptor with the Gln208Glu point mutation was used to investigate the effect of this residue on the binding of GnRH II and [D-Lys6]-GnRH II. The IC50 for GnRH II binding to the human wild-type GnRH receptor was 135.6 ± 15.8 nM, compared with 323.0 ± 33.1 nM for binding to the rat wild-type GnRH receptor (P < 0.05). The Gln208Glu human receptor mutant had an IC50 for GnRH II of 350 ± 15.5 nM, which is significantly different from the human wild-type receptor (P < 0.001) but not significantly different from the rat wild-type receptor (Fig. 1Go).


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Table 4. Comparison of Human, Mouse, and Rat GnRH Receptor Extracellular Loop (ECL) Amino Acid Sequences1

 


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Figure 1. GnRH II (open bars) and [D-Lys6]-GnRH II (filled bars) Binding to the Human Gln208 Glu Mutant GnRH Receptor Compared with Binding to the Wild-Type Human and Rat GnRH Receptors

Data are presented as mean ± SEM. IC50 values of between three and five experiments carried out in triplicate. IC50 values were calculated as described in Materials and Methods. *, Significantly different from wild-type human, P < 0.05. ***, Significantly different from wild-type human, P < 0.001.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
There is now considerable accumulated evidence that mGnRH interacts with its receptor in a ß-II' turn conformation involving residues 5–8 (7, 8, 9, 10, 11, 12, 13). This conformation appears to be conferred by interactions between Ser4 and Arg8, as well as between pGlu1 and Gly10-NH2 (13). Others have presented evidence for interactions of Arg8 with His2 and Tyr5 (9, 10, 13) contributing to the ß-II' turn conformation. An interaction of Arg8 with an acidic residue in extracellular loop 3 of the receptor is also believed to contribute to the configuration of the ligand in the folded conformation (21, 23).

D-aa6 Substitution and 6,7 {gamma}-lactam insertion further stabilize this conformation and enhance binding affinity (15, 16, 17, 24, 25). Indeed, the D-aa6 constraint can abrogate the need for the interaction between Arg8 of mGnRH and the acidic residue in extracellular loop 3 of the receptor (23). The present studies confirmed these concepts as D-Trp6, D-Ala6, and D-Lys6 substitution and 6,7 {gamma}-lactam insertion all significantly increased the binding affinity of mGnRH at the mouse GnRH receptor.

The potency of mGnRH increases with increasing hydrophobicity of the D-aa6 (24), and this is exemplified in the current study in which binding affinity successively increased in the series D-Trp6 > D-Ala6 > 6,7 {gamma}-lactam > D-Lys6. Because [D-Trp6]-GnRH has a much higher affinity than the 6,7 {gamma}-lactam analog, it appears that the substitution with D-Trp6 makes additional contributions to enhancement of affinity. Explanations for this include: possible hydrophobic interactions with the receptor; a stabilization of the ligand conformation by intramolecular hydrophobic interactions, or a reduction in flexibility due the size of these side chains; and/or a reduction in the desolvation penalty upon binding to the receptor due to the more hydrophobic nature of the ligand (12). [D-Lys6]-GnRH binds to the mouse and catfish receptors with significantly lower affinity than [6,7 {gamma}-lactam]-GnRH, implying that the basic lysine side chain is detrimental to the binding of the GnRH ligand. The hydrophilic nature of this residue may disrupt one or more of the effects described above. Additionally, the positive charge of lysine may repel the positively charged Arg8 and so affect ligand conformation. D-Arg6/D-Lys6 substitution into sGnRH, which does not contain positively charged amino acids, resulted in a much greater increase in binding affinity in the mouse, chicken, and catfish receptors. This concurs with the recorded effects of D-Arg6 substitution in sGnRH at the goldfish receptor (26, 27).

Previous studies have shown that some non-mGnRH receptors, unlike mGnRH type I receptors, are not selective for Arg8 containing ligands (20, 28). In view of the role of Arg8 in configuring the ligand, it was suggested that these receptors do not require GnRH to be configured in the ß-II' turn conformation (29). This interpretation was further supported by the observation that D-aa6 substitution did not enhance binding at the cloned chicken receptor (20) and that NMR showed the N and C termini of cGnRH I were not closely apposed (13).

However, in the present study, D-aa6 substitution clearly increased the binding affinity of mGnRH, cGnRH I, and sGnRH at mouse, chicken, and catfish receptors.

Although these observations appear to be contradictory, closer inspection reveals that the primary data concur. The interpretation that D-aa6 substitution does not enhance binding affinity at the cloned chicken receptor was based on [D-Arg6]-GnRH II having the same binding affinity as GnRH II and a GnRH analog incorporating D-Ala6 having the same affinity as mGnRH (20). The present study found a lack of enhancement for D-Arg6 substitution into GnRH II, and that D-Ala6 substitution into mGnRH produced a relatively small enhancement. Our data showing an increase in affinity resulting from D-Trp6 substitution are supported by a study testing LH releasing activity using dispersed chicken anterior pituitary cells, in which D-Trp6 analogs of cGnRH I and mGnRH were approximately 20-fold more potent than cGnRH I and mGnRH, respectively (30). Furthermore, our data showing increased binding affinity of sGnRH at non-mGnRH receptors after D-aa6 substitution are supported by two studies that showed D-Arg6 substitution in sGnRH enhanced binding affinity at the goldfish receptor (26, 27).

The NMR data show that the N and C termini of cGnRH I are not closely apposed; however, a turn conformation around Gly6 was still identified (13). Conformational energy analysis indicates that cGnRH I can adopt the ß-II' turn conformation (16). It is therefore likely that mGnRH and cGnRH I have similar conformations around Gly6, but different conformations of the termini. This may explain how these ligands have such different affinities for the same receptor.

Studies using fluorescence (10) and NMR (13) have indicated a greater flexibility of the cGnRH I ligand compared with mGnRH. This implies that the nonmammalian receptors are able to stabilize the bioactive ligand conformation, either as a result of additional receptor contact sites, or as a result of a different spatial arrangement of conserved receptor contact sites as previously proposed (20). The latter concept is supported by the observation that non-mGnRH receptors have a different conformation of extracellular loop 3 due to the altered positioning of a proline residue (31).

Most D-aa6 substitutions had limited effects on the binding affinity of GnRH II in contrast to that observed for mGnRH, cGnRH I, and sGnRH. D-Trp6 substitution, which gave the greatest increase in binding affinity of mGnRH (8.6- to 74-fold), did not substantially increase the binding affinity of GnRH II to any of the receptors (Tables 1Go and 3Go), and none of the D-aa6 substitutions substantially increased the binding affinity of GnRH II at the chicken, catfish, Xenopus II, or bullfrog III GnRH receptors. Because GnRH II binds with high affinity to these receptors and D-aa6 substitution does not substantially increase affinity, GnRH II would appear to be preconfigured in a bioactive conformation suitable for binding non-mGnRH receptors, as proposed previously (32). A more recent study also found that D-aa6 substitution did not improve the binding affinity of GnRH II at the catfish receptor, again concluding that this ligand interacts with the catfish receptor in a constrained ß-II' turn conformation (33).

Only D-Lys6 substantially increased the binding affinity of GnRH II at the human and mouse GnRH receptors. In view of the failure of other D-aa6 substitutions to increase binding affinity, we considered that D-Lys6 might provide an additional ligand-receptor interaction that does not occur with non-mGnRH receptors. A candidate interaction is between the basic D-Lys6 and an acidic residue. To address this possibility, extracellular domain acidic residues conserved in mGnRH type I receptors were screened with GnRH II and [D-Lys6]-GnRH II, using point-mutated mouse receptors. These mutations did not alter the binding affinity of either ligand (Table 2Go). Although there are other amino acid residues that can interact with a Lys side chain, we have not investigated these. Instead, we revisited the possibility that D-Lys6 substitution can contribute to the configuration of GnRH II.

Although we have evidence that GnRH II is preconfigured, its lower affinity at mGnRH type I receptors (IC50 of 128–323 nM compared with 0.75–3.7 nM at non-mGnRH receptors) suggests the interaction is not optimal. We cannot therefore rule out the possibility that D-Lys6 substitution may alter the conformation of GnRH II to improve its binding to mGnRH type I receptors.

D-Arg6 as well as D-Lys6 substantially increased the binding affinity of GnRH II at the rat GnRH receptor. This concurs with previous findings using rat pituitary membranes (34). D-Lys6 increased the binding affinity of GnRH II at the rat receptor much more (33.8-fold) than at the human (7.2-fold) and mouse receptors (8.4-fold) (Tables 1Go and 3Go). A search for amino acids different in the rat from the mouse and human revealed the presence of a glutamate residue in extracellular loop 2 of the rat in the homologous position to a glutamine residue in the human and mouse receptors. The mutation of Gln208 to Glu in the human receptor reduced the binding affinity of GnRH II to that of the rat receptor, and D-Lys6 substitution increased binding affinity by a similar amount to that found in the rat receptor. The deleterious effect of Glu208 may be due to charge repulsion between this acidic residue and acidic residues in the N-terminal region of extracellular loop 3, such as Asp293 and Glu295, causing distortion of binding site configuration and/or influencing the ease with which the ligand can interact with contact sites within the transmembrane domains. The binding affinity of [D-Lys6]-GnRH II is not significantly different between the three receptors, suggesting that D-Lys6 substitution overcomes the deleterious effect of Glu208.

In conclusion, we have obtained data in support of the concept that, as for the mGnRH type I receptors, the non-mGnRH receptors have a preference for GnRH in the folded conformation involving a ß-II' turn for residues 5–8, which is enhanced in mGnRH, cGnRH I, and sGnRH by D-aa6 substitution. In contrast, the evolutionarily conserved GnRH II ligand appears to have a preconfigured ß-II' turn that accounts for its relatively high affinity for all GnRH receptors and a failure, in most instances, of any enhancement of binding affinity with D-aa6 substitution. The surprising total conservation of GnRH II’s primary structure from bony fish to man appears to have been a product of the coordinated evolutionary selection of amino acids contributing to binding, activation, and configuration such that its structure cannot be improved by substitution with any natural amino acid at any position.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GnRH Analogs
mGnRH (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-GlyNH2), [D-Trp6]-GnRH, [D-Ala6]-GnRH, [D-Lys6]-GnRH, cGnRH I ([Gln8]-GnRH), sGnRH ([Trp7,Leu8]-GnRH), and GnRH II ([His5,Trp7,Tyr8]-GnRH) were supplied by Bachem (Saffron Walden, Essex, UK) (Table 5Go). [D-Ala6]-cGnRH I, [D-Arg6]-sGnRH, [D-Lys6]-sGnRH, [D-Trp6]-GnRH II, [D-Arg6]-GnRH II, and [D-Lys6]-GnRH II were gifts from University of Cape Town (Cape Town, South Africa). [6,7 {gamma}-Lactam]-GnRH and [6,7 {gamma}-lactam]-cGnRH I were gifts from R. Freidinger (Merck \|[amp ]\| Co., Inc., West Point, PA) and R. Roeske (Indiana University School of Medicine, Indianapolis, IN).


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Table 5. Sequences of GnRH Ligands

 
GnRH Receptor cDNA
The human (35), mouse (36), chicken (20), and Xenopus II (Troskie, B., N. Illing, and R. Millar, unpublished results) GnRH receptor cDNA constructs were gifts from Cape Town University. The rat GnRH receptor was cloned by this laboratory (37). The catfish GnRH receptor cDNA (38) was a gift from Utrecht University (Utrecht, The Netherlands). The bullfrog III GnRH receptor cDNA was a gift from Chonnam National University (Kwangju, Republic of Korea) (28). The bullfrog III receptor has greatest sequence homology for designated type II receptors (Troskie, B., N. Illing, and R. Millar, unpublished results) and should be regarded as a type II receptor. Its classification as type III was based on tissue distribution.

The mouse GnRH receptors, each having one of the conserved extracellular domain acidic residues mutated to its isosteric amide, were gifts from C. Flanagan (University of Cape Town). They were produced as described previously (21). The human receptor containing the Gln208 Glu mutation was also produced previously (39).

Cell Culture and Transfection
COS-7 cells were seeded in 100-mm2 dishes at a density of 1.2 x 106 cells per dish. Cells were maintained at 37 C, 5% CO2 in DMEM containing 10% fetal calf serum, 0.3 mg/ml glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin (Sigma-Aldrich Corp., Poole, Dorset, UK). After 24 h, the cells were transiently transfected with GnRH receptor cDNA from various species (10 µg of DNA per 100-mm2 dish) using Superfect (QIAGEN, Crawley, West Sussex, UK) according to manufacturer’s instructions (30 µl Superfect per 100-mm2 dish for 8 h). After a further 48-h incubation, cells were scraped in PBS, pelleted, and stored at -70 C.

Receptor Binding Assays
The cell pellets were homogenized in ice-cold buffer (20 mM Tris, 2 mM MgCl2, pH 7.2) and centrifuged at 15,000 rpm for 10 min at 4 C. The crude membrane pellet was then resuspended in ice-cold assay buffer (40 mM Tris, 2 mM MgCl2, 0.1% BSA, pH 7.4). Competition binding assays were carried out using radiolabeled 125I-[His5, D-Tyr6]-GnRH (~120,000 cpm/tube). The high binding affinity of this tracer compared with conventional tracers was established previously (40). The membrane suspension was incubated overnight at 4 C with labeled ligand and varying concentrations of unlabeled GnRH analogs in triplicate. The suspensions were then filtered through a membrane harvester (Brandel, St. Albans, Herts, UK) onto Whatman GF/B filter paper (Merck, Lutterworth, Leics, UK) (presoaked in assay buffer containing 0.01% polyethylenimine) and washed three times with ice-cold assay buffer. Bound radioactivity was counted using a multigamma counter [Perkin-Elmer Corp. (Wallac, Inc.), Cambridge, UK]. Maximum specific binding ranged between approximately 5,000 and 10,000 cpm/tube with nonspecific binding ranging between approximately 2,000 and 4,000 cpm/tube. No specific binding was detected with COS-7 cells transfected with vector only. Membrane concentration was varied to control for expression levels, with cells from two 100-mm2 dishes being used for each binding curve, with the exception of the human, chicken, and catfish receptors. The human and chicken receptors exhibited particularly low expression levels; therefore, four 100-mm2 dishes per curve were used. Conversely, the catfish receptor exhibited particularly high expression levels, and so one 100-mm2 dish per curve was used. Therefore, similar maximal specific radioligand binding was observed at all receptors.

Data Reduction and Statistical Analysis
Binding curves were generated by Prism graphing software (GraphPad Software, Inc., San Diego, CA) using nonlinear regression, assuming one-site competition. Significant differences in wild-type to mutant IC50 values were assessed using a two-tailed, unpaired Student’s t test with Welch’s correction (does not assume equal variances).


    ACKNOWLEDGMENTS
 
The authors would like to acknowledge the expert technical assistance of R. Sellar, N. Miller, P. Taylor, and G. Crawford. We are very grateful to R. Freidinger, R. Roeske, and R. Milton for providing GnRH analogs and to C. Flanagan and T. Ott for providing cDNA constructs.


    FOOTNOTES
 
This work was supported by the Medical Research Council.

Portions of this work were presented previously at the 83rd Annual Meeting of the Endocrine Society (32 ).

Abbreviations: cGnRH, Chicken GnRH; mGnRH, mammalian GnRH; NMR, nuclear magnetic resonance; sGnRH, salmon GnRH.

Received for publication April 30, 2002. Accepted for publication June 10, 2002.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Conn PM, Crowley Jr WF 1994 Gonadotropin-releasing hormone and its analogs. Annu Rev Med 45:391–405[CrossRef][Medline]
  2. Millar R, Lowe S, Conklin D, Pawson A, Maudsley S, Troskie B, Ott T, Millar M, Lincoln G, Sellar R, Faurholm B, Scobie G, Kuestner R, Terasawa E, Katz A 2001 A novel mammalian receptor for the evolutionarily conserved type II GnRH. Proc Natl Acad Sci USA 98:9636–9641[Abstract/Free Full Text]
  3. Millar RP 2002 Gonadotropin-releasing hormones and their receptors. In: Fauser BCJM, ed. Reproductive medicine: molecular cellular and genetic fundamentals. Lancaster, UK: Parthenon Publishing; 199–224
  4. Troskie B, Illing N, Rumbak E, Sun YM, Hapgood J, Sealfon S, Conklin D, Millar R 1998 Identification of three putative GnRH receptor subtypes in vertebrates. Gen Comp Endocrinol 112:296–302[CrossRef][Medline]
  5. White RB, Eisen JA, Kasten TL, Fernald RD 1998 Second gene for gonadotropin-releasing hormone in humans. Proc Natl Acad Sci USA 95:305–309[Abstract/Free Full Text]
  6. King JA, Millar RP 1992 Evolution of gonadotropin- releasing hormones. Trends Endocrinol Metab 3: 339–346
  7. Momany FA 1976 Conformational energy analysis of the molecule, luteinizing hormone-releasing hormone. I. Native decapeptide. J Am Chem Soc 98:2990–2996[Medline]
  8. Millar RP, Tobler CJ, King JA, Arimura A 1984 Region-specific antisera in molecular biology of neuropeptides: application in quantitation, structural characterization and metabolism of luteinizing hormone-releasing hormone. In: Soreq H, ed. Molecular biology approach to the neurosciences. New York: Wiley; 221–230
  9. Shinitzky M, Fridkin M 1976 Structural features of luliberin (luteinizing hormone-releasing factor) inferred from fluorescence measurements. Biochim Biophys Acta 434:137–143[Medline]
  10. Milton RCdL, King JA, Badminton MN, Tobler CJ, Lindsey GG, Fridkin M, Millar RP 1983 Comparative structure-activity studies on mammalian [Arg8] LH-RH and chicken [Gln8] LH-RH by fluorimetric titration. Biochem Biophys Res Commun 111:1082–1088[Medline]
  11. Guarnieri F, Weinstein H 1996 Conformational memories and the exploration of biologically relevant peptide conformations: an illustration for the gonadotropin-releasing hormone. J Am Chem Soc 118:5580–5589[CrossRef]
  12. Mezei M, Guarnieri F 1998 Computer simulation studies of the fully solvated wild-type and mutated GnRH in extended and ß-turn conformations. J Biomol Struct Dyn 16:723–732[Medline]
  13. Maliekal JC, Jackson GE, Flanagan CA, Millar RP 1997 Solution conformations of gonadotropin releasing hormone (GnRH) and [Gln(8)]GnRH. S Afr J Chem 50:217–219
  14. Rivier JE, Struthers RS, Porter J, Lahrichi SL, Jiang G, Cervini LA, Ibea M, Kirby DA, Koerber SC, Rivier CL 2000 Design of potent dicyclic (4–10/5–8) gonadotropin releasing hormone (GnRH) antagonists. J Med Chem 43:784–796[CrossRef][Medline]
  15. Monahan MW, Amoss MS, Anderson HA, Vale W 1973 Synthetic analogs of the hypothalamic luteinizing hormone releasing factor with increased agonist or antagonist properties. Biochemistry 12:4616–4620[Medline]
  16. Momany FA 1976 Conformational energy analysis of the molecule, luteinizing hormone-releasing hormone. 2. Tetrapeptide and decapeptide analogues. J Am Chem Soc 98:2996–3000[Medline]
  17. Freidinger RM, Veber DF, Perlow DS, Brooks JR, Saperstein R 1980 Bioactive conformation of luteinizing hormone-releasing hormone: evidence from a conformationally constrained analog. Science 210:656–658[Medline]
  18. Flanagan CA, Zhou W, Chi L, Yuen T, Rodic V, Robertson D, Johnson M, Holland P, Millar RP, Weinstein H, Mitchell R, Sealfon SC 1999 The functional microdomain in transmembrane helices 2 and 7 regulates expression, activation, and coupling pathways of the gonadotropin-releasing hormone receptor. J Biol Chem 274:28880–28886[Abstract/Free Full Text]
  19. Blomenröhr M, Bogerd J, Leurs R, Schulz RW, Tensen CP, Zandbergen MA, Goos HJ 1997 Differences in structure-function relations between nonmammalian and mammalian gonadotropin-releasing hormone receptors. Biochem Biophys Res Commun 238:517–522[CrossRef][Medline]
  20. Sun YM, Flanagan CA, Illing N, Ott TR, Sellar R, Fromme BJ, Hapgood J, Sharp P, Sealfon SC, Millar RP 2001 A chicken gonadotropin-releasing hormone receptor that confers agonist activity to mammalian antagonists. Identification of D-Lys(6) in the ligand and extracellular loop two of the receptor as determinants. J Biol Chem 276:7754–7761[Abstract/Free Full Text]
  21. Flanagan CA, Becker II, Davidson JS, Wakefield IK, Zhou W, Sealfon SC, Millar RP 1994 Glutamate 301 of the mouse gonadotropin-releasing hormone receptor confers specificity for arginine 8 of mammalian gonadotropin-releasing hormone. J Biol Chem 269:22636–22641[Abstract/Free Full Text]
  22. Flanagan CA, Rodic V, Konvicka K, Yuen T, Chi L, Rivier JE, Millar RP, Weinstein H, Sealfon SC 2000 Multiple interactions of the Asp(2.61(98)) side chain of the gonadotropin-releasing hormone receptor contribute differentially to ligand interaction. Biochemistry 39:8133–8141[CrossRef][Medline]
  23. Fromme BJ, Katz AA, Roeske RW, Millar RP, Flanagan CA 2001 Role of aspartate7.32(302) of the human gonadotropin-releasing hormone receptor in stabilizing a high- affinity ligand conformation. Mol Pharmacol 60:1280–1287[Abstract/Free Full Text]
  24. Karten MJ, Rivier JE 1986 Gonadotropin-releasing hormone analog design. Structure-function studies toward the development of agonists and antagonists: rationale and perspective. Endocr Rev 7:44–66[Medline]
  25. Sealfon SC, Weinstein H, Millar RP 1997 Molecular mechanisms of ligand interaction with the gonadotropin-releasing hormone receptor. Endocr Rev 18:180–205[Abstract/Free Full Text]
  26. Murthy CK, Turner RJ, Nestor JJJ, Rivier JE, Peter RE 1994 A new gonadotropin-releasing hormone (GnRH) superagonist in goldfish: influence of dialkyl-D-homoarginine at position 6 on gonadotropin-II and growth hormone release. Regul Pept 53:1–15[Medline]
  27. Illing N, Troskie BE, Nahorniak CS, Hapgood JP, Peter RE, Millar RP 1999 Two gonadotropin-releasing hormone receptor subtypes with distinct ligand selectivity and differential distribution in brain and pituitary in the goldfish (Carassius auratus). Proc Natl Acad Sci USA 96:2526–2531[Abstract/Free Full Text]
  28. Wang L, Bogerd J, Choi HS, Seong JY, Soh JM, Chun SY, Blomenröhr M, Troskie BE, Millar RP, Yu WH, McCann SM, Kwon HB 2001 Three distinct types of GnRH receptor characterized in the bullfrog. Proc Natl Acad Sci USA 98:361–366[Abstract/Free Full Text]
  29. Millar RP, King JA 1984 Structure-activity relations of LH-RH in birds. J Exp Zool 232:419–423[Medline]
  30. Millar RP, King JA 1983 Synthesis and biological activity of [D-Trp6] chicken luteinizing hormone-releasing hormone. Peptides 4:425–429[Medline]
  31. Millar RP, Troskie B, Sun YM, Ott T, Wakefield I, Myburgh D, Pawson A, Davidson JS, Flanagan C, Katz A, Hapgood J, Illing N, Weinstein H, Sealfon SC, Peter RE, Terasawa E, King JA, Plasticity in the structural and functional evolution of GnRH: a peptide for all seasons. Advances in comparative endocrinology. Proc 13th International Congress of Comparative Endocrinology, Yokohama, Japan, 1997, pp 17–27 (Scharrer-Bargmann lecture—transcript of lecture published by Monduzzi Editore S.p.A., Bologna, Italy, 1997)
  32. Pfleger KDG, Millar RP, D-Amino acid incorporation in position 6 of GnRH II does not enhance binding affinity: evidence for conformational constraint. Program of the 83rd Annual Meeting of The Endocrine Society, Denver, CO, 2001, p 550 (Abstract P3-476)
  33. Blomenröhr M, ter Laak T, Kuhne R, Beyermann M, Hund E, Bogerd J, Leurs R 2002 Chimaeric gonadotropin-releasing hormone (GnRH) peptides with improved affinity for the catfish (Clarias gariepinus) GnRH receptor. Biochem J 361:515–523[CrossRef][Medline]
  34. Millar RP, Milton RC, Follett BK, King JA 1986 Receptor binding and gonadotropin-releasing activity of a novel chicken gonadotropin-releasing hormone ([His5, Trp7, Tyr8]GnRH) and a D-Arg6 analog. Endocrinology 119:224–231[Abstract]
  35. Chi L, Zhou W, Prikhozhan A, Flanagan C, Davidson JS, Golembo M, Illing N, Millar RP, Sealfon SC 1993 Cloning and characterization of the human GnRH receptor. Mol Cell Endocrinol 91:R1–R6
  36. Tsutsumi M, Zhou W, Millar RP, Mellon PL, Roberts JL, Flanagan CA, Dong K, Gillo B, Sealfon SC 1992 Cloning and functional expression of a mouse gonadotropin- releasing hormone receptor. Mol Endocrinol 6:1163–1169[Abstract]
  37. Eidne KA, Sellar RE, Couper G, Anderson L, Taylor PL 1992 Molecular cloning and characterisation of the rat pituitary gonadotropin-releasing hormone (GnRH) receptor. Mol Cell Endocrinol 90:R5–R9
  38. Tensen C, Okuzawa K, Blomenröhr M, Rebers F, Leurs R, Bogerd J, Schulz R, Goos H 1997 Distinct efficacies for two endogenous ligands on a single cognate gonadoliberin receptor. Eur J Biochem 243:134–140[Abstract]
  39. Ott TR, Troskie BE, Roeske RW, Illing N, Flanagan CA, Millar RP 2002 Two mutations in extracellular loop 2 of the human GnRH receptor convert an antagonist to an agonist. Mol Endocrinol 16:1079–1088[Abstract/Free Full Text]
  40. Flanagan CA, Fromme BJ, Davidson JS, Millar RP 1998 A high affinity gonadotropin-releasing hormone (GnRH) tracer, radioiodinated at position 6, facilitates analysis of mutant GnRH receptors. Endocrinology 139:4115–4119[Abstract/Free Full Text]