Residues within Transmembrane Helices 2 and 5 of the Human Gonadotropin-Releasing Hormone Receptor Contribute to Agonist and Antagonist Binding

Silke H. Hoffmann, Ton ter Laak1, Ronald Kühne, Helmut Reiländer and Thomas Beckers

Department of Cancer Research (S.H.H., T.B.) ASTA Medica AG D-60314 Frankfurt/Main, Germany
Department of Molecular Membrane Biology (S.H.H., H.R.) Max-Planck-Institute of Biophysics D-60528 Frankfurt/Main, Germany
Institute of Molecular Pharmacology (T.t.L., R.K.) D-10315 Berlin, Germany


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To understand the ligand binding properties of the human GnRH receptor (hGnRH-R), 24 site-specific mutants within transmembrane helices (TMH) 1, 2, and 5 and the extracellular loop 2 (E2) were generated. These mutants were analyzed by using a functional reporter gene assay, monitoring receptor signaling via adenylate cyclase to a cAMP-responsive element fused to Photinus pyralis luciferase. The functional behavior of 14 receptor mutants, capable of G-protein coupling and signaling, was studied in detail with different well described agonistic and antagonistic peptide ligands. Furthermore, the binding constants were determined in displacement binding experiments with the antagonist [125I]Cetrorelix.

The substitution of residues K36, Q204, W205, H207, Q208, F210, F213, F216, and S217 for alanine had no or only a marginal effect on ligand binding and signaling. In contrast, substitution of N87, E90, D98, R179, W206, Y211, F214, and T215 for alanine resulted in receptor proteins neither capable of ligand binding nor signal transduction. Within those mutants affecting ligand binding and signaling to various degrees, W101A, N102A, and N212Q differentiate between agonists and antagonists. Thus, in addition to N102 already described, the residues W101 in TMH2 and N212 in TMH5 are important for the architecture of the ligand-binding pocket. Based on the experimental data, three-dimensional models for binding of the superagonist D-Trp6-GnRH (Triptorelin) and the antagonist Cetrorelix to the hGnRH-R are proposed. Both decapeptidic ligands are bound to the receptor in a bent conformation with distinct interactions within the binding pocket formed by all TMHs, E2, and E3. The antagonist Cetrorelix with bulky hydrophobic N-terminal amino acids interacts with quite different receptor residues, a hint at the failure to induce an active, G protein-coupling receptor conformation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The decapeptide GnRH (or LHRH), discovered by Schally and Guillemin and co-workers (1 2 ), provides the pivotal force driving the human reproductive axis and therefore is of fundamental medical and biological importance. GnRH is synthesized in the gonadotropic cells of the hypothalamus and released in a pulsatile manner to stimulate LH and FSH release from the pituitary (for review see Ref. 3 ). Superagonistic derivatives of GnRH like D-Trp6-GnRH/Triptorelin (Decapeptyl) (4 ) or D-Ser(tBu)6-GnRH/Buserelin (Suprecur) (5 ) and potent antagonists like Cetrorelix (Cetrotide; ASTA Medica AG, Frankfurt, Germany) (6 ) were developed for treatment of sex hormone-dependent diseases as precocious puberty, endometriosis and prostate cancer or for in vitro fertilization (reviewed in Refs. 7 8 9 ). Conformation-function studies suggested that the central tetrapeptide of GnRH adopts a ß-II turn as the dominant conformation to maintain the proper spatial arrangement of the terminal fragments, presumably participating in docking to the receptor (10 11 ). However, such a conformation has never been observed experimentally.

The GnRH receptor (GnRH-R) expressed on gonadotropic cells of the anterior pituitary gland belongs to the family of rhodopsin-like G protein-coupled receptors (GPCRs), characterized by seven putative transmembrane helices (TMH 1–7) (reviewed in Refs. 12 13 ). The GnRH-R was cloned from several mammalian (human, marmoset monkey, mice, rat, sheep, bovine) and nonmammalian species (catfish, goldfish, chicken) (reviewed in Ref. 13 ). The mammalian GnRH-R shares several unique features including the absence of a cytoplasmic C-terminal tail, replacement of Y by S140 in the conserved DRY sequence in TMH3 and the reciprocal interchange of two residues highly conserved among GPCRs, N87 in TMH2, and D319 in TMH71. Studies on GnRH-R signal transduction revealed a versatile and dynamic network of temporally segregated responses (reviewed in Refs. 14 15 ). The GnRH-R couples to Gq/G11 but may as well interact with Gi and Gs in rat pituitary cells (16 17 ). Heterologously expressed GnRH-R also couples to adenylate cyclase in GH3 rat pituitary tumor cells (18 19 ) or insect cells (20 ), emphasizing the promiscuity of the GnRH-R as a function of the availability of G proteins in the microenvironment of the test cell.

Caused by the poor solubility of GPCRs and difficulties in obtaining crystals suitable for x-ray structural analysis, existing structural information of G protein-coupled receptors is limited to rhodopsin (21 22 23 ). Thus, to date, generating site-specific mutants of GPCRs is the only experimental approach to study structure-function relationships (24 ). Concurring knowledge for the GnRH-R has been accumulated during the last years (13 25 26 ). The relevance of N87 (TMH2) and D318 (TMH7) for the murine and rat GnRH-R structure has been shown by several groups (27 28 29 ). The N87D mutant was unable to bind agonists, whereas the D318N mutant showed ligand binding but signaling was severely impaired (27 29 ). Reciprocal interchange in the N87D/D318N mutant restored ligand binding but not signal transduction. A recent study by Mitchell et al. (30 ) revealed that D318N, as well as the N87D/D318N mutant of murine GnRH-R, was unable to activate phospholipase D in an ADP-ribosylation factor (ARF) independent way (30 ). These results indicate that N87 and D319 and therefore TMH2 and 7 are located in close proximity and are important for receptor signaling.

With L58, L73, S74, and L80 as part of intracellular loop I (I1) (31 ), L147 within the conserved DRYXXV/ISSPL motif of GPCRs I2 (32 ), R262 in I3 (33 ), and Y322 from the DPLIY motif in TMH7 (34 ), several other residues affecting signal transduction have been identified. Mutants in I1 abolished or severely affected signaling by cAMP, but not inositol phosphate (IP) responses (31 ). Thus the I1 loop might be important for binding and activating Gs proteins. The R262Q mutant, which was characterized by wild-type (wt) agonist binding affinity but a 10-fold reduced stimulation of IP production in heterologous expression experiments, was detected as a GnRH-R gene mutant in a family with idiopathic hypogonadotropic hypogonadotropism (IHH) (33 36 ). The DRS138–140 motif at the cytoplasmic border of TMH3 was also subject to mutagenesis. The D138A mutant abolished ligand binding and signal transduction, whereas the D138N and D138E mutants were characterized by almost wt affinity for agonists, yet signaling was affected (37 38 ). The I143A mutant showed impaired receptor activation, presumably by solvation of R139 in the cytoplasmic aqueous environment. Proper positioning of R139 achieved by I143 allows interaction with, e.g. D138 and is supposed to be a prerequisite for receptor activation. Therefore, in the proposed model, R139 is involved in the transition between the inactive and active receptor conformation (37 ).

Until now, only N102 (TMH2), K121 (TMH3), and D302 (E3/TMH7) have been identified as being important for ligand binding. The interaction of Arg8 of mammalian GnRH with E301 of the murine GnRH-R (D302 in human) was proposed by Flanagan et al. (39 ). The mutants K121L, D, or Q abolished ligand binding, but antagonist binding to the K121Q mutant was unaffected and therefore an interaction of K121 with amino-terminal residues was proposed (40 ). The N102A mutant described by Davidson et al. (41 ) differentiates between agonists with glycineamide and ethylamide C termini, suggesting that N102 forms a hydrogen bond with the C-terminal amide moiety. The authors assumed that N102 is located proximal to D302 and K121, which are part of the ligand-binding pocket formed by TMH 1, 2, 3, and 7.

To facilitate the functional characterization of hGnRH-R mutants and for having a reasonable throughput of ligands, we have established a modified reporter gene assay based on GnRH-R signaling via adenylate cyclase (42 ). Here we applied this methodology for the functional characterization of 24 site-specific mutants of the human GnRH-R. Receptor residues in TMH1, TMH2 with focus to the E2-loop, and TMH5 were selected to detect those residues involved in ligand binding. In most cases, residues were subjected to alanine substitution (Ala-scan mutagenesis). After selecting cell lines with stable expression of hGnRH-R mutants, functional properties as well as binding affinities for the antagonist Cetrorelix were analyzed in detail. In this report we present two models of the human GnRH-R with the agonistic and antagonistic ligands D-Trp6-GnRH and Cetrorelix, respectively, docked into the GnRH binding pocket. A deep hydrophobic binding pocket is formed by TMHs 1, 2, 3, and 7 as described recently (41 ), but also with a major contribution of residues within TMH 5 and 6. The residue N212 in TMH 5 was found to be important for agonist binding and for the stabilization of the antagonist binding pocket. By comparing the functional and binding activity of agonists and antagonists, W101 and N102 in TMH2 were found to be important for agonist, but much less so for antagonist binding.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Quantitative Analysis of GnRH-R Signal Transduction by Using a Luciferase Reporter Gene-Based Functional Assay
A functional assay was established for quantitative analysis of GnRH-R activation by agonists as described (42 ). The assay is based on a clonal murine L-cell line harboring a Photinus pyralis luciferase reporter gene under control of the cAMP responsive element, which was fused to a cytomegalovirus minimal promoter. The wt hGnRH-R was stably transfected into this reporter cell line. The induction of luciferase gene expression by D-Trp6-GnRH was analyzed by Northern blotting (Fig. 1AGo). The concentration-dependent induction of luciferase activity by GnRH or D-Trp6-GnRH was quantified in cell lysates and, as depicted in Fig. 1BGo, showed the activation of the cAMP signaling pathway by heterologously expressed hGnRH-R. As expected, the antagonist Cetrorelix did not effect luciferase activity in unstimulated cells, but concentration-dependently inhibited the GnRH-R activation by D-Trp6-GnRH (Northern blot, Fig. 1CGo, and concentration-response curve in Fig. 1BGo) (42 44 ). In addition to GnRH, D-Trp6-GnRH, and Cetrorelix, the GnRH derivatives des-Gly10-ProNHEt9-GnRH, des-Gly10-D-Trp6-Pro-NHEt9-GnRH, D-Ala6-GnRH, and des-Gly10-D-Ala6-Pro-NHEt9-GnRH, as well as the antagonist Antarelix, were included in these initial assay validation experiments. Antarelix differs from Cetrorelix only at position 6 and 8 [D-homocitrulline/Hci6 and isopropyllysine/Lys(iPr)8 instead of D-citrulline/Cit and arginine in Cetrorelix]. In all experiments, quantification of luciferase reporter gene allowed a highly reproducible EC/IC50 determination correlating with binding affinity (Table 1Go).



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Figure 1. Induction of Reporter Gene Expression by GnRH or D-Trp6 GnRH and Inhibition by Cetrorelix

The reporter gene cell line expressing the wt GnRH-R was stimulated with 1 nM D-Trp6-GnRH for various times and transcriptional induction of the luciferase gene was analyzed by Northern blotting (A). Cellular luciferase activity was concentration-dependently induced by GnRH or D-Trp6-GnRH (EC50 = 2.0 and 0.74 nM, respectively) or antagonized by Cetrorelix (IC50 = 0.94 nM, panel B). The antagonistic activity in the functional assay correlated with luciferase gene expression, as was analyzed by Northern blotting of RNA isolated from cells treated with different concentrations of Cetrorelix before stimulation with D-Trp6-GnRH (C). The very low basal transcriptional activity of the CRE3-CMVmin promoter controlling the luciferase gene can be seen in untreated cells (lanes 1 and 2). In this experiment the PDE inhibitor IBMX was used to enhance the D-Trp6-GnRH-induced signal (lane 4).

 

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Table 1. Summary of Data Derived from Functional and Displacement Binding Experiments for hGnRH-R Mutants with Signal Transduction Capacity

 
Residues of the Human GnRH-R Exchanged by Site-Directed Mutagenesis
The residues N87, E90, D98, and N102 already known from the literature, as well as new residues in TMH1 (K36), TMH2 (W101), the E2-Loop (R179, Q204, W205, W206, H207, Q208, F210), and TMH5 (Y211, N212, F213, F214, T215, F216, S217), were selected as targets for site-specific mutagenesis (Fig. 2Go). A strong emphasis was put on residues in the outer part of TMH5 and neighboring residues in the E2 loop, which were mutagenized to alanine systematically from residues 204–217 (except residue 209 which is A in the wt GnRH-R). Expression plasmids harboring the wt or mutant (mt) hGnRH-R genes were stably transfected into the reporter gene cell line. After selection, the cell pools were immediately used for the functional and displacement binding assays.



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Figure 2. Schematic View of the Human GnRH-R and Mutated Residues

The human GnRH-R (51 52 ) is shown in a simplified model. Residues exchanged in this study by site-directed mutagenesis are highlighted. The single N-glycosylation site of the human receptor (N18) (64 ) and disulfide bonds between C14–C200 and C114 –C196 (54 60 ) are also shown.

 
Functional Characterization of hGnRH-R Mutants Described in the Literature
For reasons of comparison the GnRH-R mutants that had already been described in the literature were studied first. The amino acids N87, E90, D98, and N102 were mutagenized to A. The EC50 values for each transfectant were calculated from concentration-response curves. The results summarizing the functional and displacement binding data of all mutants in this study are presented in Table 1Go. To facilitate the data interpretation, the EC/IC50, KD, and Bmax values obtained for the mt hGnRH-Rs were divided by that of the wt hGnRH-R. The resulting activity, affinity, or expression indices indicated, for example, the apparent decrease in potency, meaning the influence of a receptor mutation on ligand binding (affinity) or receptor signaling (activity).

The A mutants of residues N87 and D98 showed neither receptor signaling nor ligand binding. Data published by several groups for the N87D and D98N mutants described no or decreased activity, respectively (27 28 30 39 ). Our finding that D98A is completely inactive stresses the importance of residue D98 for GnRH-R function. Contrary to a normal activity for the E90Q mutant claimed by Flanagan et al. (39 ), our result revealed that the mutant E90A was inactive.

The N102A mutant was capable of coupling to G protein but with significant differences between the various agonists (Table 1Go and Fig. 5Go, A–C). With an EC50 of 530 ± 100 nM for GnRH with the glycineamide C terminus the activity index was 240 (Fig. 5AGo). In contrast, the activity index of the agonist des-Gly10-Pro9-NHEt-GnRH showing an EC50 of 29 ± 4 nM was calculated to 62 (Fig. 5BGo). Similar results were obtained with D-Ala6-GnRH and des-Gly10-D-Ala6-Pro-NHEt9-GnRH and to some extent also with D-Trp6-GnRH (Fig. 5CGo) and des-Gly10-D-Trp6-Pro-NHEt9-GnRH. A new feature of the N102 residue is that it proved to be of minor importance for antagonist binding. The activities of Cetrorelix and Antarelix with an IC50 of 5.4 ± 0.9 nM (activity index 4.9) and 8.2 ± 0.8 nM (activity index 5.9), respectively, were close to that of the wt receptor. For comparison, the activities of the conformationally constrained agonists were significantly more affected (Table 1Go and Fig. 5Go, C and D).




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Figure 5. Functional Behavior of the N102A, W101A, and N212A/Q Mutants

Mutants N102A, W101A in TMH2, and N212A/Q in TMH5 were analyzed for GnRH, des-Gly10-ProNHEt9-GnRH, or D-Trp6-GnRH-induced receptor activation (A, B, C, E, and G) and inhibition of D-Trp6-GnRH induced activation by Cetrorelix (D, F, and H). For GnRH the EC50 is shifted from 2.0 ± 0.2 nM for the wt hGnRH-R to 639 ± 80 nM for the N102A mutant (A); for des-Gly10-ProNHEt9-GnRH from 0.31 ± 0.04 nM to 32.6 ± 8.2 nM (B); for D-Trp6-GnRH from 0.71 ± 0.11 nM to 14.8 ± 3.7 nM (C). For Cetrorelix the IC50 is shifted from 0.94 ± 0.11 nM for wt hGnRH-R to 4.7 ± 0.7 nM for the N102A mutant (D). For the W101A mutant the EC50 of GnRH is shifted to 7.1 ± 0.5 µM (E), for Cetrorelix the IC50 is shifted to 40 ± 7.5 nM (F). In case of the N212A mutant, the EC50 for GnRH is shifted to 54 ± 13 nM (G), the IC50 for Cetrorelix to 64 ± 12 nM (H). In contrast, the N212Q mutant differentiates between agonist and antagonists as depicted by a shift for GnRH to EC50 = 37 ± 7 nM (G) but nearly wt activity for Cetrorelix with IC50= 2.7 ± 0.5 nM (H). Similar results were obtained by testing other agonistic and antagonistic derivatives (Table 1Go).

 
Functional Analysis of New hGnRH-R Mutants within TMH1, TMH2, E2, and TMH5
Several amino acid residues of the human GnRH-R with focus on residues in E2 and TMH5 were selected for mutagenesis and characterized in functional and displacement binding experiments (Fig. 2Go). Concentration-response curves for all hGnRH-R mutants capable of signal transduction were typically sigmoid shaped curves, as depicted in Fig. 3Go for GnRH (A) or the antagonist Cetrorelix (B). The wt hGnRH-R was characterized by an EC50 of 2.2 ± 0.2 nM for GnRH; the EC50 values obtained for the hGnRH-R mutants ranged from 0.92 nM (F210A) to 6.3 µM (W101A). For Cetrorelix, an IC50 = 1.1 ± 0.2 nM was calculated for inhibition of D-Trp6-GnRH induced signaling via the wt GnRH-R, whereas for the mutant receptors IC50 values ranged from 0.7 ± 0.2 nM (F210A) to 53 ± 16 nM (N212A). All hGnRH-R mutants displayed sigmoid-shaped [125I]-Cetrorelix binding curves (data not shown), and the dissociation constant (KD) as well as the Bmax values as calculated from the binding data were included in Table 1Go. In Fig. 4Go the binding affinities (KD) were plotted against the antagonistic potency (IC50) for wt and mutant GnRH-R and depicted a linear relationship. The alanine substitution in the E90, D98, R179, W206, Y211, F214, and T215 mutants, which exhibited no signaling activity, also failed in [125I]-Cetrorelix binding.



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Figure 3. Functional Characteristics of hGnRH-R Mutants

The hGnRH-R mutants in TMH1 (K36A), TMH2 (W101A, N102A), the E2-Loop (Q204 A, W205A, H207A, Q208A, F210A), and TMH5 (N212A/Q, F213A/Y, F216A, S217A) were analyzed for GnRH-induced receptor activation (A) and inhibition of D-Trp6-GnRH-induced activation by Cetrorelix (B). The normalized concentration-response curves of typical experiments are shown, emphasizing the sigmoid-shaped curves.

 


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Figure 4. Correlation of Binding Affinity and Antagonistic Potency of hGnRH-R Mutants

The binding affinity (KD) of Cetrorelix as determined in displacement binding experiments with [125I]Cetrorelix was correlated with the antagonistic potency as determined in functional reporter gene assays. The hGnRH-R mutants as shown in Fig. 2Go were included using vital GnRH-receptor expressing cells. The displacement binding and functional assay were performed under comparable physiological conditions. In the functional assay, Cetrorelix treatment was performed with a 15 min preincubation step. Cellular luciferase activity was determined in cell lysates 6 h after stimulation with D-Trp6-GnRH. Cell-bound [125I]Cetrorelix was quantified 1 h after incubation with tracer.

 
Mutants with a discriminating behavior toward agonists and antagonists were W101A and N212A/Q (Table 1Go and Fig. 5Go). The W101A mutant had a strong effect on receptor activation and ligand binding, reflected by an activity index for agonists of 730 to 6000 (EC50 from 0.55 to 6.30 µM; Fig. 5EGo for GnRH). Surprisingly, the activities for the antagonists Cetrorelix and Antarelix with activity indices of 39 and 37, respectively, (IC50 = 43 and 52 nM; Table 1Go and Fig. 5FGo for Cetrorelix) were decreased significantly less. This correlated with the binding affinity for Cetrorelix, which was decreased by a factor of 23 (KD = 4.8 nM). In analogy to the N102A mutant described above, a small but significant discrimination between agonists with glycineamide and ethylamide C-termini was detected (Table 1Go). In contrast to the N102A mutation, agonists with a glycineamide C terminus-like GnRH (activity index 2900) and D-Trp6-GnRH (activity index 730) were more potent than the respective ethylamide variants des-Gly10-Pro-NHEt9-GnRH (activity index 6000) and des-Gly10-D-Trp6-Pro-NHEt9-GnRH (activity index 1670).

The amino acid residue N212 located in TMH5 was mutated to A and Q. Both mutations and here especially the N212A mutant displayed a marked increase of the EC50 values for agonists (Table 1Go and Fig. 5GGo for GnRH). This result suggested that N212 is located within the binding pocket and contributes to agonist binding, which is further supported by the receptor model (Fig. 6CGo and Discussion). Yet a surprising difference between the N212A and N212Q mutants was detected during testing with antagonist (Table 1Go and Fig. 5HGo for Cetrorelix). The N212A mutant exhibited a significant effect on the antagonistic potency of Antarelix and Cetrorelix (activity indices of 40 and 48, respectively), which also correlated with the decreased binding affinity determined for Cetrorelix. Thus, the N212A mutant exhibited no discrimination between agonists and antagonists. However, the N212Q mutant displayed almost wt behavior in respect to Cetrorelix binding and signal inhibition.



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Figure 6. Molecular Models for D-Trp6 GnRH and Cetrorelix Binding to the hGnRH-R

A and B, Top view of the receptor complex with bound ligand. The position of D-Trp6-GnRH (A) or Cetrorelix (B) in the hGnRH-R transmembrane bundle, represented as cylinders, is shown using the view from the extracellular site. C, Stereo view on the molecular model of D-Trp6-GnRH bound to the hGnRH-R. D-Trp6-GnRH is present in a tight turn conformation placing the amino acid D-Trp6 between TMH3 and 6 away from the ligand binding site. In this model and in agreement with the experimental data, the D-Trp6 side chain of Triptorelin lays toward the extracellular side close to the N-terminal part of the receptor. The first three N-terminal amino acids of D-Trp6-GnRH bind into the receptor pocket between TMH3, 4, 5, and 6. The positively charged Arg8 makes an interaction with D302 at the top of TMH7. The C-terminal glycine amide (GLD10) interacts with N102 and D98 at the top of TMH2 and places Pro9 relatively deep in the hydrophobic pocket between TMH1, 2, and 7. Receptor residues interacting directly with D-Trp6-GnRH are green, residues having effects on the receptor structure are cyan, and residues showing no effects in our mutagenesis study or not yet examined experimentally are white. D, Stereo view on the molecular model of the antagonist Cetrorelix bound to hGnRHR. Cetrorelix is present in a tight-turn conformation placing the amino acid side chain of D-Cit6 between TMH3 and 6 away from the ligand-binding site toward the extracellular space. Cetrorelix contains the three bulky amino acids D-naphtylalanine (Nal1), D-p-Cl-phenylalanine (Clp2), and D-pyridylalanine (Pal3). These hydrophobic residues fill the receptor pocket between TMH 3, 4, 5, and 6 and are not able to interact with K121. At the C terminus, Cetrorelix contains D-Ala10-NH2 (Ald10), which appears to prohibit W101 from interacting with the decapeptide backbone and makes Cetrorelix binding more insensitive against the N102A substitution. Green labels indicate receptor residues that have a direct interaction with Cetrorelix or are important for the stabilization of the binding pocket; cyan labels, residues stabilizing the receptor structure; white labels, residues showing no effects in our mutagenesis study.

 
A Model for D-Trp6-GnRH and Cetrorelix Binding to the hGnRH-R
The superagonist D-Trp6-GnRH and the antagonist Cetrorelix were docked into the hGnRH-R model using a molecular dynamics protocol with carefully designed range distance restraint functions (see Materials and Methods). D-Trp6-GnRH was forced to enter the hGnRH-R pocket based on the following experimental information on receptor mutants of the present work and published data: 1) D302 interacts with Arg8 of GnRH as was revealed by the study of the murine GnRH-R by Flanagan et al. (39 ); 2) the hGnRH-R mutant N102A showed a much greater decrease in binding affinity for Gly10-NH2 than for N-ethylamide ligands, suggesting that the C-terminal Gly10-NH2 residue interacts with N102 (Table 1Go) (41 ); 3) K121 can be mutated to R but not to Q, L, or D without loss of receptor function, suggesting that K121 participates in a salt bridge (40 ); and finally 4) photoaffinity labeling studies have shown that the position of D-Trp6 of Triptorelin is located close to C14 in the GnRH-R (54 ). Since there are strong similarities between the decapeptides D-Trp6-GnRH and Cetrorelix, we assumed that Arg8 of Cetrorelix interacts with D302, D-Ala10-NH2 is placed near N102, and the space-filling D-Cit6 side chain has a similar orientation as D-Trp6 in Triptorelin. Two three-dimensional models of the hGnRH-R were constructed at a molecular level and were examined for accordance with the experimental data. After 40 stimulated annealing (SA) runs with the restraints as mentioned above, the models were selected that agreed best with the mutagenesis experiments of the present study. The models as shown in Fig. 6Go are discussed in more detail in the figure legend and together with the functional data of the various hGnRH-R mutants in the following section.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The GnRH-R system is of outstanding importance for basic biology and endocrinology as well as for treatment of sex hormone-dependent diseases (3 12 ). At the present time superagonists are widely used for treatment of gynecological disorders and hormone-dependent cancers. Cetrorelix (Cetrotide) (6 ) was the first antagonist that became available for in vitro fertilization. The pharmacological relevance of the GnRH-R led to an increasing interest in the elucidation of the three-dimensional receptor structure, a prerequisite for the rational design of novel, potent GnRH analogs. Recently, the first nonpeptidic GnRH-R antagonist with oral bioavailability was described (56 ) and future research will focus on these low molecular mass compounds. To date, detailed structural information on the arrangement of the seven putative transmembranal segments present in all GPCRs is still missing. Thus, studies to characterize the binding pocket of GPCRs have relied on indirect approaches, such as functional effects of site-specific receptor mutants and computational three-dimensional molecular models of ligand-receptor complexes (24, 57 and database on http://www-grap.fagmed.uit.no/GRAP/homepage.html].

Various mutants of the GnRH-R affecting ligand binding or G protein coupling and signal transduction have been described (for review see Refs. 13 25 ). The labor-intensive functional assays based on IP production and detection limits with standard radioligand assays kept mutants or ligands to be tested to small numbers. Therefore, no detailed model for ligand binding and almost no data concerning the architecture of the antagonist binding pocket are currently available. In the present study multiple receptor mutants were characterized by using a panel of peptidic agonistic and antagonistic GnRH derivatives. A high-throughput functional assay was applied to generate a comprehensive set of data needed to propose and validate computational models for D-Trp6-GnRH and Cetrorelix binding to the human GnRH-R. With focus on TMH5 and E2, 20 residues of the hGnRH-R were exchanged by site-directed mutagenesis to A and in a few cases to closely related residues (N->Q, F->Y). The functional assay as described (42 ) was adopted for characterization of the mutant hGnRH-Rs in stably transfected cells. In the antagonist experiments, D-Trp6-GnRH was used for receptor activation in a concentration of 2x EC50 of the respective receptor mutant. By doing so, comparable and practicable experimental conditions were generated to compensate for the very different activities of receptor mutants. In addition, all mutants were characterized by displacement binding experiments with intact cells and [125I] Cetrorelix (44 ). This radioligand is superior to agonists like [125I]-Triptorelin in terms of high binding affinity and >=80% of tracer capable for specific receptor association. Thus, it was possible to determine dissociation constants for receptor mutants with decreased expression or binding affinity, a major problem in many studies (58 ). There was a very good correlation of the binding affinity KD and the antagonistic potency IC50 of Cetrorelix (Fig. 4Go), and those mutants without signaling activity also did not bind the antagonist. Therefore, we assume that the mutations affected ligand binding, while signal transduction is only affected as a consequence. From the displacement binding assays Bmax values for the different mutant receptor proteins exposed on the cell surface were calculated (Table 1Go, last column). Relative to wt hGnRH-R production, the mutant receptor proteins were produced between 150% (F210A) and 17% (W101A). This can be explained either by differences in transfection efficiency and/or mutant receptor protein synthesis, processing, or transport. As has been discussed recently, the EC50 could depend on the number of receptors expressed on the cell surface (40 59 ). From our data we conclude that this is unlikely, because 1) weakly or strongly expressed mutants like H207A, W205A, or F210A had wt activity, 2) the shape of all dose-response curves was identical, and 3) receptor affinity and antagonistic potency did correlate. In addition the maximal effect observed ín model experiments to decrease transiently overexpressed GnRH-R number artificially was 10-fold (40 ).

One can deduce from the mutational analysis that the residues K36 (TMH1), Q204, W205, H207 Q208, F210 (E2), and F213 and S217 (TMH5) played a minor role in binding of the peptidic ligands. In accordance to the receptor model, their activity and affinity were identical or close to that of the wt receptor (Fig. 6Go and Table 1Go). The S217R mutant has been described recently in a family with IHH together with mutations in E1 (Q106R) and I3 (R262Q) (36 ). The S217R receptor mutant alone was incapable of ligand binding and signaling. Since S217 is conserved in all mammalian GnRH-Rs except mouse (G216), it is likely that only small, uncharged residues are tolerated at this position. In the receptor model, S217 is pointing toward the lipid membrane; thus, the positively charged guanidinium group of the S217R mutant most probably hinders a proper membrane integration whereas the alanine exchange is tolerated.

Mutations of residues N87, E90, D98, R179, W206, Y211, F214, and T215 to A resulted in a complete loss of ligand binding as well as signal transduction. One has to keep in mind that this can be explained by 1) destruction of the overall receptor structure and/or ligand binding pocket, 2) failure of proper integration into the membrane and therefore loss of surface expression, or 3) synthesis of a misfolded protein targeted to the proteasome. Mutants at the loci N87, E90, and D98 have already been published for the murine (27 28 30 ), rat (29 ), and catfish GnRH-R (61 ). For mammalian GnRH-R, the N87D mutation was inactive, and N87 was considered to form a salt bridge with D319 in TMH7, which is important for the structural integrity of the receptor. Therefore, it was not surprising to find that the N87A mutant of hGnRH-R neither binds ligand nor undergoes agonist-induced receptor activation and signaling.

The E90Q mutant of the murine GnRH-R showed wt behavior considering agonist binding and signal transduction (39 ). In the receptor model E90 forms a salt bridge with K121 in TMH3, which probably is essential for receptor structure and explains the loss of function due to the alanine mutation (Fig. 6Go). Interaction with K121 via hydrogen bonds is also possible for a glutamine side chain, which explains the wt behavior of the E90Q mutant. In our modeling studies we have experienced that K121 was able to interact with pGlu1 or His2 of agonists without interfering with residual receptor-ligand interactions. Taking into consideration the calculated interaction energies, the pGlu1-K121 interaction was slightly favored (data not shown).

In comparison to the wt hGnRH-R and depending on the ligands studied, signaling and ligand binding capacity of the W101A, N102A (TMH2), N212A/Q, and F216Y (TMH5) mutants was affected quite differently. The W101A mutant displayed the most pronounced shift in agonist-induced signal transduction published so far (Fig. 5EGo). In the model, W101 is involved in binding via a hydrogen bond with the Leu7 backbone oxygen of D-Trp6-GnRH (Fig. 6CGo). In contrast, this interaction was not observed in the Cetrorelix receptor binding model which is in agreement with the smaller effect in case of antagonists (Fig. 5FGo). Therefore, we assume that W101 might be important for the proper formation of the binding pocket around the C-termini in case of antagonists.

Mutants of N102 were already described recently: the N102Q mutation neither affected neither ligand binding, signaling, nor receptor production (26 41 ). In contrast, the N102A mutation caused a marked bias between the C termini of agonists. The activity of GnRH and the agonists with a glycineamide C terminus was reduced about 27- to 220-fold, whereas the activity of agonists with an ethylamide C terminus was much less affected (Table 1Go and Ref. 41 ). The authors assumed that N102 was important for binding of the C terminus of agonists. In our experiments, we were able to confirm this assumption for agonists. Nevertheless, in contrast to agonists, the antagonistic potency and binding affinity of Cetrorelix was only slightly reduced. According to our receptor models, N102 most probably is directly involved in ligand binding of the glycineamide C terminus of D-Trp6-GnRH and the D-alanylamide C terminus of Cetrorelix via a hydrogen bond. The D-Ala side chain stabilizes the orientation of the C terminus of Cetrorelix and, therefore, antagonist binding is less affected by this mutation.

As was deduced from the data of the N212A/Q mutants that exhibited a different behavior for agonists and antagonists, the residue N212 in TMH5 directly contributes to the architecture of the ligand binding pocket. In our model, N212 is involved in the binding of the D-Trp6-GnRH backbone (His2 = O) via a hydrogen bond. In the case of Cetrorelix, the N212 residue appears not to be directly involved in binding of the antagonist, but seems to be more important for stabilizing the binding pocket through hydrogen bonds with W206. These differences in agonist/antagonist binding are supported by the result that the N212Q mutant did not affect antagonist binding, whereas agonist binding was severely reduced most probably by steric hindrance.

Although the F216A mutant showed wt activity, the F216Y mutant affected agonist as well as antagonist binding. In our model, the side chain of F216 is near the ligand binding pocket and not directly involved in ligand interaction. It is likely that at this position a hydrophobic side chain is needed and that the hydrophilic phenolic side chain of tyrosine distorts the receptor conformation due to an interaction with the Y283.

By comparing the EC50 values of agonistic ligands of the W101A, N102A, N212A/Q, and F216Y receptor mutants, a common theme became apparent. In contrast to GnRH, signaling of these GnRH-R mutants after induction with the structurally constrained analog D-Trp6-GnRH and to some extent D-Ala6-GnRH was significantly less affected. For example, D-Trp6-GnRH and GnRH have an activity index of 27 and 240, respectively, for the N102A mutant and of 3.2 and 19, respectively, for the F216Y mutant. Similar results have been published for the N102A mutant by Davidson et al. (41 ) and the D301Q murine receptor mutant by Flanagan et al. (39 ). The antagonistic potency and binding affinity of Cetrorelix or Antarelix is also much less affected by these receptor mutants. Therefore, it is reasonable to postulate that the initial steps of ligand binding, i.e. the docking of superagonistic and antagonistic GnRH analogs might be quite similar. In contrast to GnRH, these ligands are conformationally constrained due to the incorporation of bulky and D-configurated amino acid side chains (13 ). GnRH has more conformational freedom and, therefore, needs an induced fit for receptor binding and activation. In the case of agonists, receptor-ligand interactions are required to induce an active receptor complex. Such interactions are of less importance for the constrained analogs. Although there are similarities between D-Trp6-GnRH and Cetrorelix, the functional data and the receptor models highlight the differences. Many interactions of the N-terminal residues of D-Trp6-GnRH (pGlu1-His2-Trp3) are not present in the hydrophobic and bulky N-terminal sequence D-Nal1-D-pClPh-Ala2-D-Pal3 of Cetrorelix. In addition, the Cetrorelix model lacks the K121-pGlu1 or alternatively the K121-His2 interaction. Here K121 interacts only with E90 in TMH2. Since the N-terminal parts of the agonistic GnRH analogs are predominantly responsible for receptor activation (13 ), this gives a hint to the antagonistic function of analogs like Cetrorelix. Certainly, the models as proposed have to be refined and confirmed by additional receptor mutants, especially to gain a comprehensive understanding of antagonist binding and function on a molecular level. Nevertheless, the receptor mutants and the computational models as presented are very useful tools for defining putative binding sites for small, peptidomimetic antagonistic ligands like T-98475 (56 ) and, subsequently, the design of derivatives with a higher potency or specificity. Based on cell lines with stable expression of hGnRH-R receptor mutants and a sensitive reporter system for quantification of receptor activation at hand, it is feasible to comprehensively characterize new receptor mutants and peptidomimetic compounds.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Recombinant DNA Procedures and Site-Specific Mutagenesis
Unless otherwise specified, standard molecular biological protocols described by Ausubel et al. (43 ) were applied. Site-specific mutagenesis was performed essentially following the manual of CLONTECH Laboratories, Inc. (Palo Alto, CA) Transformer Site-directed Mutagenesis Kit. The hGnRH-R cDNA was cloned into the pSBC1 plasmid as has been described elsewhere (44 ). The Trans Oligo AlwNI/SpeI (CLONTECH Laboratories, Inc. manual) was chosen as selection primer. The mutagenesis primers were designed as follows:

K36A: 5'-CTTGTCTGGAGCGATCCGAGTG-3', N87A: 5'-GACCTTAGCCGCCCTGTTG GAG-3', D98A: 5'-CATGCCACTGGCTGGGATGTGG-3', W101A: 5'- GGATGGGATGGC CAACATTACAGTC-3', N102A: 5'-GGGATGTGGGCCATTACAGTCC-3', R179A: 5'-CAGTTATACA TCTTCGCGATGATTCATCTAG-3', Q204A: 5'-CTGCAGTTTTTCAGCCTGGTGG-CATC-3', W205A: 5'-CAGTTTTTCACAAGCTTGGCATCAAG-C-3', W206A: 5'-GTTTTTCACAATGGGCCCATC AAGCA-TTTTATAAC-3', W206Y: 5'-GTTTTTCACAATGGTACCATCA-AGCATTTTATAAC-3', W205A/W206A: 5'-CAGTTTTTCACAA-GCTGCGCATCAAGCATTTTATAAC-3', H207A: 5'-CACAAT-GGTGGGCCCAAGCATTTTATAAC-3', Q208A: 5'-CAATGGT-GGCATGCAGCATTTTATAAC-3', F210A: 5'-GCATCAAG-CGGCCTATAACTTTTTCACC-3', Y211A: 5'-GCA TCAAG-CATTCGCGAACTTTTTCACC-3', N212A: 5'-CATCAAGCAT-TTTACGCGTTTTTCACCTTCAGC-3', N212Q: 5'-CATCA-AGCATTTTATCAATTTTT-CACCTTCAG-3', F213A: 5'-CAAG-CATTTTATAACGCGTTCACCTTCAG-3', F214A: 5'-GCATTT-TATAACTTCGCG ACCTTCAGCTG-3', T215A: 5'-CTAGGA-ATTTGGGCCTGGTTTGATCC 3'; F216A: 5'-CTTT TTCAC-CGCGAGCTGCCTCTTC-3', F216Y: 5'-CTTTTTCACCTACAG-CTGCCTC-3', S217A: 5'-CTTTTTCACCTTCGCATGCCTC-TTCATC-3', C218A: 5'-ACCTTCAGCCTCTCTTCATCATC 3'.

By AseI/NotI fragment shuffling between the pSBC1-hGnRHR derivatives and pSBC2-SEAP, dicistronic expression vectors pSBC mt hGnRHR/IRES/SEAP were generated (44 ). Mutations were verified by restriction analysis and DNA sequencing, using the dideoxy chain termination method according to Sanger.

Mammalian Cell Culture, Stable Transfection, and Selection of Cell Clones
Murine thymidine kinase-deficient L cells (LTK-) and transfectants thereof were cultured at 37 C, 5% CO2 in DMEM supplemented with 10% inactivated FCS (FCSi), penicillin/streptomycin, and glutamine. LTK- cells were transfected by calcium phosphate/DNA coprecipitation (45 ) with plasmids pSBC(CRE3)CMVmin-Luc containing the firefly luciferase cDNA under control of the cAMP-response element (CRE) fused to the cytomegalovirus (CMV) minimal promoter (our unpublished data) and pSV2 PAC containing the puromycin-N-acetyl-transferase gene (46 ). After selection (culture medium supplemented with 5 µg/ml puromycin), single-cell clones were isolated by limited dilution and about 200 clones were analyzed for expression of the Luc reporter gene after stimulation with forskolin. Four clones with optimal signal-noise ratio and intermediate or high signal after forskolin stimulation were selected and stably supertransfected with the pSBC-wt hGnRHR/IRES/SEAP plasmid and pAG60 containing the amino-glycoside phosphotransferase gene (47 ). After selection (culture medium supplemented with 800 µg/ml G418/Life Technologies, Inc., Gaithersburg, MD), cell pools were analyzed for luciferase expression after stimulation with D-Trp6-GnRH. Clonal cell line 122 showed superior stimulation characteristics (75,000 vs. 6,000 relative light units of unstimulated cells) and, therefore, was chosen as the reporter cell line for stable supertransfection with the hGnRH-R mutants. The cell pools obtained after selection with G418 were used for analysis of signal transduction and ligand binding.

Isolation of RNA and Northern Blot Analysis
Subconfluent cultures of cells were stimulated with 1 nM D-Trp6-GnRH and 500 µM 3-isobutyl-1-methylxanthine (IBMX) and harvested after 0, 30, 60, 120, 240, and 360 min by adding lysis buffer (4 M guanidinium thiocyanate, 0.5% (wt/vol) sodium lauryl sarcosinate, 285 mM ß-mercaptoethanol, 25 mM sodium citrate, pH 7) directly to the culture dishes. Preparation of total RNA, electrophoretic separation, transfer to the Hybond N+ nylon membrane, hybridization with [32P]dCTP random labeled probes, and finally stringent washing was performed as has been described previously (48 ). For experiments with the antagonist, 0.1–1000 nM Cetrorelix was added 1 h before stimulation with 1 nM D-Trp6-GnRH/500 µM IBMX, and cells were lysed 6 h after stimulation. For hybridization, a cDNA probe of Photinus pyralis luciferase (EcoRI/HindIII fragment from pSBC2 Luc (49 ) was used.

Agonistic and Antagonistic GnRH Analogs, Cell Treatment, and Reporter Gene Assay
Cetrorelix/D-20761 (Ac-D-Nap-Ala1-D-ClPh-Ala2-D-Pyr-Ala3-Ser4-Tyr5-D-Cit6-Leu7-Arg8-Pro9-D-Ala10-NH2) and Antarelix/D-23234 (Ac-D-Nap-Ala1-D-ClPh-Ala2-D-Pyr-Ala3-Ser4-Tyr5-D-Hci6-Leu7-Lys(iPr)8-Pro9-D-Ala10-NH2) were synthesized at ASTA Medica AG as described previously (6 ). The agonistic peptides GnRH, des-Gly10-Pro-NHEt9-GnRH, D-Trp6-GnRH, Des-Gly10-D-Trp6-Pro-NHEt9-GnRH, D-Ala6-GnRH, des-Gly10-D-Ala6-Pro-NHEt9-GnRH were purchased from Bachem Biochemica GmbH (Heidelberg, Germany). All peptides with exception of Cetrorelix were dissolved in water at 1 mM final concentration and stored in siliconized polypropylene tubes at -20 C. Cetrorelix was dissolved in 0.01 N CH3COOH.

For functional analysis of the receptor mutants 1 x 104 cells per well were cultivated for 24 h in 96-well microtiter plates using DMEM with supplements and 10% (vol/vol) FCSi. Subsequently cells were stimulated for 6 h with the respective agonist. For signal enhancement, a phosphodiesterase (PDE) inhibitor (IBMX or the PDE4 specific inhibitor Rolipram) was added. Cells were lysed for quantification of cellular Luc activity. Cetrorelix and Antarelix were added 15 min before the stimulation of cells with D-Trp6-GnRH. For stimulation a variable D-Trp6-GnRH concentration of 2x EC50 was used, dependent on the GnRH-R mutant tested. The luciferase reporter gene assay was performed as described (42 ). Calculation of the EC50 and IC50 values was done by nonlinear regression analysis using the Hill model (program EDX 2.0, our unpublished data) and mean values ± SD (sample standard deviation {varsigma}n-1) from at least two independent experiments are shown.

Radioligand Binding Assay
For receptor binding studies, [125I]Cetrorelix was prepared and used as a tracer that exhibited about 80% of peptide capable for specific receptor association. The binding assay was performed on intact cells under physiological conditions essentially as described (44 ). For displacement binding assays, 1 x 106 cells in 100 µl were incubated with {approx}225 pM [125I]Cetrorelix (specific activity 5–10 x 105 dpm/pmol) and different concentrations of unlabeled Cetrorelix as a competitor for 1 h at 37 C. Binding affinity (KD) and cell surface receptor expression (receptor concentration Bmax) were calculated from the displacement binding data by using the EBDA/Ligand analysis program (Biosoft V3.0) (50 ) and mean values ± SD (sample SD {varsigma}n-1) from independent experiments are shown.

Molecular Modeling Experiments
The starting structure for the model of the GnRH-R transmembrane domains was built from the sequence of the hGnRH-R (Swiss-Prot P30968) (51 52 ) using the {alpha}-carbon template of the transmembrane helices of rhodopsin (53 ). To obtain a complete model, the extra- and intracellular loops were added using the loop search option in SYBYL6.4. Two disulfide bridges (C14-C200 and C114-C196) reported by Davidson et al. (54 ) were included in the model. According to earlier studies on the stability of transmembrane bundles in molecular dynamics simulations (55 ), we used positively charged arginine and lysine residues and negatively charged glutamate and aspartate residues in all calculations. To obtain better three-dimensional structures for the intra- and extracellular loop regions of the hGnRH-R, a simulated annealing (SA) method was performed to sample a large number of different conformations using the AMBER 4.1 force field. In 25 SA runs the hGnRH-R was solvated with explicit water molecules and the TMH domains were restrained to maintain the template (55 ). The hGnRH-R model with intra- and extracellular loop structures having the lowest potential energy was used for further modeling studies. In comparison to the rhodopsin template structure some of the TMHs were extended as a result of the SA calculations (TMH boundaries: Lys36-Lys62, Leu73-Ile103, Leu112-Arg145, Val155-Ile181, Asn212-Val241, Arg260-Trp291, His306-Leu328). The rhodopsin template represents an arbitrary transmembrane bundle of a GPCR in the inactive state and does not reflect receptor-specific conformational changes such as proline kinks. To simulate such effects, an AMBER4.1 molecular dynamics simulation in vacuo was performed in a next step, fixing the arrangement of the seven-helix bundle by a set of distance restraints between the centers of three C{alpha}-atoms in each TMH (TMH1: Thr51, Phe52, Asn53; TMH2: Val94, Met95, Pro96; TMH3: Tyr126, Ala127, Pro128; TMH4: Ala171, Gly172, Pro173; TMH5: Ile221, Ile222, Pro223; TMH6: Trp280, Thr281, Pro282; TMH7: Phe318, Asp319, Pro320). This allows rotations of the TMHs around their own axis and enables helix kinks near prolines.

D-Trp6-GnRH was docked into the putative agonist binding site during 150 psec of AMBER4.1 molecular dynamics simulation in vacuo starting from about 30–40 Å outside the hGnRH-R pocket (55 ). The mass centers of interacting amino acid residues (pGlu1 with K121, Arg8 with D302, and Gly10-NH2 with N102) were restrained to gradually approach a distance smaller than 3–5Å or a distance smaller than 8Å (Trp6 with C14). During the simulation the TMHs were stabilized by restraints on all helix hydrogen bonds and the center of mass restraints described previously. The GnRH-R model as obtained was used to generate different D-Trp6-GnRH binding modes within the receptor binding site with SA method (40 runs, AMBER4.1, in vacuo, heating up to 1500 K in 5 psec and then slowly cooling down to 0 K between 25 psec and 50 psec). All SA runs were performed using the same set of restraints (distance restraints between pGlu1 with K121, Arg8 with D302, and Gly10-NH2 with N102; harmonic restraints of 1 kcal mol-1 Å-2 on all C{alpha}-atoms of the TMHs to fix the TM regions; distance restraints between the mass centers of the extra- and intracellular loops to avoid unfolding of loop regions during SA; torsional restraints to fix chirality of all C{alpha}-atoms).

In the case of Cetrorelix, SA runs were performed starting with the docked D-Trp6-GnRH-hGnRH-R complex in which the five distinct residues were mutated to those of Cetrorelix (pGlu1 to D-Nal1; His2 to D-pClPhe2; Trp3 to D-Pal3; D-Trp6 to D-Cit6; Gly10-NH2 to D-Ala10-NH2) using the SYBYL mutate option and only two biases for receptor-ligand interaction during the 40 SA runs (Arg8 with D302 and D-Ala10-NH2 with N102). The restraints to fix the receptor structure were identical to these described for the D-Trp6-GnRH-binding study.

After AMBER 4.1 force field minimization, the obtained receptor binding models for both D-Trp6-GnRH and Cetrorelix were ranked according to the calculated ligand binding energies. Models having the highest ligand binding energies were selected, and the influence of the mutations presented in this study on the ligand binding energy was calculated for each of these models. The binding models showing the best agreement between experimental data and calculated ligand binding energies were selected.


    ACKNOWLEDGMENTS
 
We acknowledge the excellent assistance of S. Hoevelmann, S. Moka, P. Schmidt, and E. Uloth. We also thank Dr. M. Bernd for synthesis of antagonists and Dr. E. Polymeropoulos for helpful discussions. The ligand [125I]Cetrorelix was prepared and kindly provided by Dr. G. P. McGregor, Philipps-Universität, Marburg, Germany.


    FOOTNOTES
 
Address requests for reprints to: Dr. Thomas Beckers, Department of Cancer Research, ASTA Medica AG, Weismuellerstrasse 45, Frankfurt am Main, Germany D-60314.

This work was supported by the Federal Ministry of Research, Education, Science and Technology (BMBF), Grant 0310697A.

1 Present address: Vrije Universiteit Amsterdam, Division of Chemistry, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. Back

Received for publication January 18, 2000. Revision received March 6, 2000. Accepted for publication March 21, 2000.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Schally AV, Nair RMG, Redding TW, Arimura A 1971 Isolation of the luteinizing hormone and follicle-stimulating hormone-releasing hormone from porcine hypothalami. J Biol Chem 246:7230–7236[Abstract/Free Full Text]
  2. Burgus R, Butcher M, Amoss M, Ling N, Monahan MW, Rivier J, Fellows T, Blackwell R Vale W, Guillemin R 1972 Primary structure of ovine hypothalamic luteinizing hormone-releasing factor (LRF). Proc Natl Acad Sci USA 69:278–285[Abstract]
  3. Evans JJ 1999 Modulation of gonadotropin levels by peptides acting at the anterior pituitary gland. Endocr Rev 20:46–67[Abstract/Free Full Text]
  4. Canales ES, Montvelinsky H, Fonseca ME, Zarate A, Kastin AJ, Coy DH, Schally AV 1980 The use of a potent stimulatory LHRH analog (D-Trp6-LHRH) in the induction of ovulation. Int J Fertil 25:193–197[Medline]
  5. Mansfield MJ, Beardsworth DE, Loughlin JS, Crawford JD, Bode HH, Rivier J, Vale W, Kushner DC, Crigler JF, Crowley WF 1983 Long-term treatment of central precocious puberty with a long-acting analogue of luteinizing hormone-releasing hormone. N Engl J Med 309:1286–1292[Abstract]
  6. Kutscher B, Bernd M, Beckers T, Polymeropoulos EE, Engel J 1997 Chemistry and molecular biology in the search for new LH-RH antagonists. Angewandte Chemie 109:2240–2254
  7. Conn PM, Crowley WF 1991 Gonadotropin-releasing hormone and its analogues. N Engl J Med 324:93–103[Medline]
  8. Emons G, Schally AV 1994 The use of luteinizing hormone-releasing hormone agonists and antagonists in gynaecological cancers. Hum Reprod 9:1364–1379[Abstract]
  9. Weinbauer GF, Nieschlag E 1992 LH-RH antagonists: state of the art and future perspectives. In: Höffgen K (ed) Peptides in Oncology I — LH-RH Agonists and Antagonists. Springer Verlag, Berlin, vol 124:113–136
  10. Nikiforovich FV, Marshall GR 1993 Conformation-function relationships in LHRH analogs. Int J Pept Prot Res 42:171–193
  11. Karten MJ, Rivier JE 1986 Gonadotropin-releasing hormone analog design. Structure function studies towards development of agonists and antagonists: rational and perspective. Endocr Rev 7:44–66[Medline]
  12. Stojilkovic SS, Reinhart J, Catt KJ 1994 Gonadotropin-releasing hormone receptors: structure and signal transduction pathways. Endocr Rev 15:462–499[Medline]
  13. 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]
  14. Stanislaus D, Pinter JH, Janovick JA, Conn PM 1998a Mechanisms mediating multiple physiological responses to gonadotropin-releasing hormone. Mol Cell Endocrinol 144:1–10
  15. Naor Z, Harris D, Shacham S 1998 Mechanism of GnRH receptor signaling: combinatorial cross-talk of Ca2+ and protein kinase C. Front Neuroendocrinol 19:1–19[CrossRef][Medline]
  16. Stanislaus D, Janovick JA, Brothers S, Conn PM 1997 Regulation of Gq/G11{alpha} by the GnRH receptor. Mol Endocrinol 11:738–746[Abstract/Free Full Text]
  17. Stanislaus D, Ponder S, Ji TH, Conn PM 1998b Gonadotropin-releasing hormone receptor couples to multiple G proteins in rat gonadotrophs and in GH3 cells: evidence from palmitoylation and overexpression of G proteins. Biol Reprod 59:579–586
  18. Kuphal D, Janovick JA, Kaiser UB, Chin WW, Conn PM 1994 Stable transfection of GH3 cells with rat gonadotropin-releasing hormone receptor complementary deoxyribonucleic acid results in expression of a receptor coupled to cyclic adenosine 3',5'-monophosphate-dependent prolactin release via a G-protein. Endocrinology 135:315–320[Abstract]
  19. Stanislaus D, Arora, V, Awara WM, Conn PM 1996 Biphasic action of cyclic adenosine 3',5'-monophosphate in gonadotropin-releasing hormone (GnRH) analog-stimulated hormone release from GH3 cells stably transfected with GnRH receptor complementary deoxyribonucleic acid. Endocrinology 137:1025–1031[Abstract]
  20. Delahaye R, Manna PR, Bérault A, Berreur-Bonnenfant J, Berreur P, Counis R 1997 Rat gonadotropin-releasing hormone receptor expressed in insect cells induces activation of adenylyl cyclase. Mol Cell Endocrinol 135:119–127[CrossRef][Medline]
  21. Schertler GF, Villa C, Herderson R 1993 Projections structure of rhodopsin. Nature 362:770–772[CrossRef][Medline]
  22. Unger VM, Schertler GFX 1995 Low resolution structure of bovine rhodopsin determined by electron cryo-microscopy. Biophys J 68:1776–1786[Abstract]
  23. Unger VM, Hargrave PA, Baldwin JM, Schertler GF 1997 Arrangement of rhodopsin transmembrane {alpha}-helices. Nature 389:203–206[CrossRef][Medline]
  24. Schwartz TW 1994 Locating ligand-binding sites in 7TM receptors by protein engineering. Curr Opin Biotechnol 5:434–444[Medline]
  25. Flanagan CA, Millar RP, Illing N 1997 Advances in understanding gonadotropin-releasing hormone receptor structure and ligand interactions. Rev Reprod 2:113–120[Abstract/Free Full Text]
  26. Davidson JS, Flanagan CA, Becker II, Illing N, Sealfon SC, Millar RP 1994 Molecular function of the gonadotropin-releasing hormone receptor: insights from site-directed mutagenesis. Mol Cell Endocrinol 100:9–14[CrossRef][Medline]
  27. Zhou W, Flanagan C, Ballesteros JA, Konvicka K, Davidson JS, Weinstein H, Millar RP, Sealfon SC 1994 A reciprocal mutation supports helix 2 and helix 7 proximity in the gonadotropin-releasing hormone receptor. Mol Pharmacol 45:165–170[Abstract]
  28. Awara WM, Guo CH, Conn PM 1996 Effects of Asn318 and Asp87Asn318 mutations on signal transduction by the gonadotropin-releasing hormone receptor and receptor regulation. Endocrinology 137:655–662[Abstract]
  29. Cook JV, Faccenda E, Anderson J, Couper GG, Eidne KA, Taylor PL 1993 Effects of Asn87 and Asp318 mutations on ligand binding and signal transduction in the rat GnRH receptor. J Mol Endocrinol 139:R1–R4
  30. Mitchell R, McCulloch D, Lutz E, Johnson M, MacKenzie C, Fennell M, Fink G, Zhou W, Sealfon SC 1998 Rhodopsin-family receptors associate with small G proteins to activate phospholipase D. Nature 392:411–414[CrossRef][Medline]
  31. Arora KK, Krsmanovic LZ, Mores N, O’Farrell H, Catt KJ 1998 Mediation of cyclic AMP signaling by the first intracellular loop of the gonadotropin-releasing hormone receptor. J Biol Chem 273:25581–25586[Abstract/Free Full Text]
  32. Arora KK, Sakai A, Catt KJ 1995 Effects of second intracellular loop mutations on signal transduction and internalization of the gonadotropin-releasing hormone receptor. J Biol Chem 270:22820–22826[Abstract/Free Full Text]
  33. Layman LC, Cohen DP, Jin M, Xie J, Li Z, Reindollar RH, Bolbolan S, Bick DP, Sherins RR, Duck LW, Musgrove LC, Sellers JC, Neill JD 1998 Mutations in gonadotropin-releasing hormone receptor gene cause hypogonadotropic hypogonadism. Nat Genet 8:14–15
  34. Arora KK, Cheng Z, Catt KJ 1996 Dependence of agonist activation on an aromatic moiety in the DPLIY motif of the gonadotropin-releasing hormone receptor. Mol Endocrinol 10:979–986[Abstract]
  35. Arora KK, Chung HO, Catt KJ 1999 Influence of a species-specific extracellular amino acid on expression and function of the human gonadotropin-releasing hormone receptor. Mol Endocrinol 13:890–896[Abstract/Free Full Text]
  36. de Roux N, Young J, Brailly-Tabard S, Misrahi M, Milgrom E, Schaison G 1999 The same molecular defects of the gonadotropin-releasing hormone receptor determine a variable degree of hypogonadism in affected kindred. J Clin Endocrinol Metab 84:567–572[Abstract/Free Full Text]
  37. Ballesteros J, Kitanovic S, Guarnieri F, Davies P, Fromme BJ, Konvicka K, Chi L, Millar RP, Davidson JS, Weinstein H, Sealfon SC 1998 Functional microdomains in G-protein-coupled receptors. The conserved arginine-cage motif in the gonadotropin-releasing hormone receptor. J Biol Chem 273:10445–10453[Abstract/Free Full Text]
  38. Arora KK, Cheng Z, Catt KJ 1997 Mutations of the conserved DRS motif in the second intracellular loop of the gonadotropin-releasing hormone receptor affect expression, activation, and internalization. Mol Endocrinol 11:1203–1212[Abstract/Free Full Text]
  39. 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]
  40. Zhou W, Rodic V, Kitanovic S, Flanagan CA, Chi L, Weinstein H, Maayani S, Millar RP, Sealfon SC 1995 A locus of the gonadotropin-releasing hormone receptor that differentiates agonist and antagonist binding sites. J Biol Chem 270:18853–18857[Abstract/Free Full Text]
  41. Davidson JS, McArdle CA, Davies P, Elario, R, Flanagan CA, Millar RP 1996 Asn102 of the gonadotropin-releasing hormone receptor is a critical determinant of potency for agonists containing C-terminal glycineamide. J Biol Chem 271:15510–15514[Abstract/Free Full Text]
  42. Beckers T, Reiländer H, Hilgard P 1997 Characterization of gonadotropin-releasing hormone analogs based on a sensitive cellular luciferase reporter gene assay. Anal Biochem 251:17–23[CrossRef][Medline]
  43. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K 1989 Current Protocols in Molecular Biology. John Wiley & Sons Inc., New York
  44. Beckers T, Marheineke K, Reiländer H, Hilgard P 1995 Selection and characterization of mammalian cell lines with stable over-expression of human pituitary receptors for gonadoliberin. Eur J Biochem 231:535–543[Abstract]
  45. Wigler M, Sweet R, Sim GK, Wold B, Pellicer A, Lacy E, Maniatis T, Silverstein S, Axel R 1979 Transformation of mammalian cells with genes from procaryotes and eucaryotes. Cell 16:777–785[Medline]
  46. Vara J, Portela A, Ortin J, Jimenez A 1986 Expression in mammalian cells of a gene from Streptomyces alboniger conferring puromycin resistance. Nucleic Acids Res 14:4617–4624[Abstract]
  47. Colbére-Garapin F, Horodniceanu F, Kourilsky P, Garapin AC 1981 A new dominant hybrid selective marker for higher eukaryotic cells. J Mol Biol 150:1–14[Medline]
  48. Beckers T, Schmidt P, Hilgard P 1994 Highly sensitive northern hybridization of rare mRNA using a positively charged nylon membrane. Biotechniques 16:1075–1078
  49. DeWet JR, Wood KV, DeLuca M, Helinski DR, Subramani S 1987 Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol 7:25–37
  50. McPherson GA 1985 Analysis of radioligand binding experiments: a collection of computer programs for the IBM PC. J Pharmacol Methods 14:213–228[CrossRef][Medline]
  51. Kakar SS, Musgrove LC, Devor DC, Sellers JC, Neill JD 1992 Cloning, sequencing and expressing of human gonadotropin-releasing hormone (GnRH) receptor. Biochem Biophys Res Commun 189:289–295[Medline]
  52. Chi L, Zhou W, Prikhozan A, Flanagan D, Davidson KJS, Golembo M, Illing N, Millar RP, Sealfon SC 1993 Cloning and characterization of the human gonadotropin-releasing hormone receptor. Mol Cell Endocrinol 91:R1–R6
  53. Baldwin JM, Schertler GFX, Unger VM 1997 An {alpha}-carbon template for the transmembrane helices in the rhodopsin family of G-protein-coupled receptors. J Mol Biol 272:144–154[CrossRef][Medline]
  54. Davidson JS, Assefa D, Pawson A, Davies, P, Hapgood J, Becker I Flanagan C, Roeske R, Millar RP 1997 Irreversible activation of the gonadotropin-releasing hormone receptor by photoaffiniity crosslinking: localization of attachment site to cys residue in N-terminal segment. Biochemistry 36:2881–12889
  55. Ter Laak AM, Kuehne R 1999 Bacteriorhodopsin in a periodic boundary water-vacuum-water box as an example towards stable molecular dynamics simulations of G-protein coupled receptors. Receptors Channels 6:295–308[Medline]
  56. Cho N, Harada M, Imaeda T, Imada T, Matsumoto H, Hayase Y, Sasaki S, Furuya S, Suzuki N, Okubo S, Ogi K, Endo S, Onda H, Fujino M 1998 Discovery of a novel, potent, and orally active nonpeptide antagonist of the human luteinizing hormone-releasing hormone (LHRH) receptor. J Med Chem 41:4190–4195[CrossRef][Medline]
  57. Kristiansen K, Dahl SG, Edvardsen O 1996 A database of mutants and effects of site-directed mutagenesis experiments on G protein-coupled receptors. Proteins 26:1–94[CrossRef][Medline]
  58. 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]
  59. Kenakin T 1993 Stimulus-response mechanisms. In: Pharmacologic Analysis of Drug-Receptor Interaction, ed 2. Raven Press, New York, pp 39–68
  60. Cook JV, Eidne KA 1997 An intramolecular disulfide bond between conserved extracellular cysteines in the gonadotropin-releasing hormone receptor is essential for binding and activation. Endocrinology 138:2800–2806[Abstract/Free Full Text]
  61. 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]
  62. Kaiser UB, Zhao D, Cardona GR, Chin WW 1992 Isolation and characterization of cDNAs encoding the rat pituitary gonadotropin-releasing hormone receptor. Biochem Biophys Res Commun 189:1645–1652[Medline]
  63. Reinhart J, Mertz LM, Catt KJ 1992 Molecular cloning and expression of cDNA encoding the murine gonadotropin-releasing hormone receptor. J Biol Chem 267:21281–21284[Abstract/Free Full Text]
  64. Davidson JS, Flanagan CA, Zhou W, Becker II, Elario R, Emeran W, Sealfon SC, Millar RP 1995 Identification of N-glycosylation sites in the gonadotropin-releasing hormone receptor: role in receptor expression but not ligand binding. Mol Cell Endocrinol 107:241–245[CrossRef][Medline]