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.
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ABSTRACT |
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INTRODUCTION |
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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 58 (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 67. 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 -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 58, 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.
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RESULTS |
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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 1).
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 2). 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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DISCUSSION |
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D-aa6 Substitution and 6,7 -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
-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 -lactam > D-Lys6. Because [D-Trp6]-GnRH has a much higher affinity than the 6,7
-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
-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 1 and 3
), 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 2). 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 128323 nM compared with 0.753.7 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 1 and 3
). 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 58, 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 IIs 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.
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MATERIALS AND METHODS |
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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 manufacturers 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 Students t test with Welchs correction (does not assume equal variances).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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