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Originally published In Press as doi:10.1074/jbc.M404474200 on May 21, 2004

J. Biol. Chem., Vol. 279, Issue 33, 34115-34122, August 13, 2004
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Species Selectivity of Nonpeptide Antagonists of the Gonadotropinreleasing Hormone Receptor Is Determined by Residues in Extracellular Loops II and III and the Amino Terminus*

Greg J. Reinhart{ddagger}, Qiu Xie{ddagger}, Xin-Jun Liu§, Yun-Fei Zhu¶, Jun Fan||, Chen Chen¶, and R. Scott Struthers{ddagger}**

From the Departments of {ddagger}Endocrinology, §Peptide Chemistry, Medicinal Chemistry, and ||Molecular Biology, Neurocrine Biosciences Inc., San Diego, California 92121

Received for publication, April 22, 2004 , and in revised form, May 21, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Efforts to develop orally available gonadotropin-releasing hormone (GnRH) receptor antagonists have led to the discovery of several classes of potent nonpeptide antagonists. Here we investigated molecular interactions of three classes of nonpeptide antagonists with human, rat, and macaque GnRH receptors. Although all are high affinity ligands of the human receptor (Ki <5 nM), these compounds show reduced affinity for the macaque receptor and bind only weakly (Ki >1 µM) to the rat receptor. To identify residues responsible for this selectivity, a series of chimeric receptors and mutant receptors was constructed and evaluated for nonpeptide binding. Surprisingly, 4 key residues located in the amino terminus (Met-24) and extracellular loops II (Ser-203, Gln-208) and III (Leu-300) of the GnRH receptor appear to be primarily responsible for species-selective binding. Comparisons of reciprocal mutations suggest that these may not be direct contacts but rather may be involved in organizing extracellular portions of the receptor. These data are novel because most previous reports of residues involved in binding of nonpeptide ligands to peptide-activated G protein-coupled receptors, including the GnRH receptor as well as mono-amine receptors, have identified binding sites in the transmembrane regions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Gonadotropin-releasing hormone (GnRH)1 is a linear decapeptide amide, pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, that regulates the reproductive endocrine axis in both male and female vertebrates (1, 2). The GnRH receptor (Gn-RHR) is a member of the G protein-coupled receptor superfamily (3). Its activation stimulates phospholipase C and phosphatidyl inositol turnover by coupling to the GTP-binding proteins Gq and G11 (4). Stimulation of pituitary GnRH receptors by hypothalamic GnRH released into the pituitary portal circulation results in secretion of the gonadotropins, luteinizing hormone, and follicle-stimulating hormone into the peripheral circulation. The gonadotropins in turn regulate the production of steroids and gametes by the gonads. Receptors for GnRH have been cloned from a wide range of species (5), and numerous studies have been reported elucidating key residues in the receptor responsible for peptide binding and signal transduction (5).

Peptide drugs that inhibit the action of GnRH have proven useful in regulating the hypothalamic-pituitary-gonadal axis and are now routinely used for ablation of gonadal steroids. This approach has proven effective in the management of endometriosis, prostate cancer, uterine fibroids, breast cancer, and other conditions, including infertility (6). However, the requirement for daily injection or implantation of long acting depots has prompted a number of groups to attempt to develop orally active, nonpeptide antagonists (for a review, see Ref. 7). These efforts have been hindered in part by the species selectivity that is observed for most nonpeptide GnRH antagonists. Cho et al. (8) reported synthesis of T-98475, a nonpeptide GnRH antagonist with high affinity to the human GnRH receptor. However, this compound was 20-fold less potent against the receptor from the cynomolgus macaque (Macaca fascicularis) and 300-fold less potent against the rat receptor. Similarly, an unrelated nonpeptide GnRH antagonist (9) was found to be highly potent against the human but 50- to 300-fold less potent at the dog receptor. We have reported several nonpeptide antagonist series with remarkable selectivity for the human receptor compared with the rat receptor (10–13). These observations are somewhat surprising given that, with one or two exceptions (14, 15), similar degrees of species selectivity have not been reported for the thousands of peptide GnRH analogs that have been synthesized by numerous groups (16).

The rat GnRH receptor is highly homologous (87%) to the human receptor, whereas the receptor cloned from the rhesus macaque (Macaca mulatta) contains only 8 substitutions of the 328 amino acids in the human sequence. Therefore, to identify the molecular basis of this species selectivity and identify residues involved in nonpeptide ligand binding to the GnRH receptor, we have investigated residues responsible for the selectivity of several nonpeptide GnRH antagonists for the human receptor over receptors from rat and macaque. Systematic mutagenesis of extracellular and transmembrane residues was used to identify critical nonpeptide contacts responsible for human/macaque selectivity. Chimeric rat/human receptors were employed to localize regions responsible for selectivity, and individual residues were then identified by systematic mutagenesis. Here we report that species selectivity for three families of nonpeptide GnRH antagonists is determined by residues in the amino terminus and extracellular loops II and III.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Rhesus pituitary tissue was a generous gift from Sierra Biomedical. [D-LEU6,NMeLeu7,Pro9-NEt]GnRH was synthesized by solid phase methods. The synthesis of nonpeptide compounds is reported elsewhere (13, 17).2

Preparation of GnRH Radioligand—The GnRH analog [D-Leu6,NMeLeu7,Pro9-NEt]GnRH was labeled by the chloramine-T method. 1 mCi of Na125I was added to 10 µg of peptide in 20 µl of 0.5 M sodium phosphate buffer, pH 7.6, followed by 22.5 µg of chloramine-T in 15 µl of 0.05 M sodium phosphate buffer. The mixture was vortexed for 20 s. The reaction was stopped by the addition of 60 µg of sodium metabisulfite in 30 µl of 0.05 M sodium phosphate buffer. The free iodine was removed by passing the reaction mixture through a C-8 Sep-Pak cartridge (Millipore Corp., Milford, MA). The peptide was eluted with a small volume of 80% acetonitrile/water. The recovered labeled peptide was further purified by reverse phase high pressure liquid chromatography on a Vydac C-18 analytical column (The Separations Group, Hesperia, CA) on a Beckman 334 gradient HPLC system using a gradient of acetonitrile in 0.1% trifluoroacetic acid. The purified radioactive peptide was stored in 0.1% bovine serum albumin/20% acetonitrile/0.1% trifluoroacetic acid at –80 °C and can be used for up to 4 weeks.

Cloning Rhesus Macaque Receptor—Total RNA was isolated from rhesus macaque (M. mulatta) pituitary tissue using the RNeasy mini kit (Qiagen) and first strand cDNA made with the cDNA synthesis kit from Invitrogen. The full coding sequence of the GnRHR was amplified with PCR primers corresponding to the human GnRHR cDNA sequence. The 5'-primer (5'-catgatctcgagaagctctgtcctgggaaaatatg-3') contained a XhoI restriction site followed by 17 bases of human 5'-untranslated sequence ending with the start codon. The 3'-primer (5'-gacgaattcatatgacttcttgtgtagtctatcaa-3') corresponded to 29 bp of human 3'-untranslated sequence immediately following the stop codon of the human receptor, followed by an EcoRI I restriction site. A band of ~1kb was amplified, isolated, and cloned into the expression vector pCDNA3.1(–) (Invitrogen). Six independent clones were isolated and sequenced on an automated sequencer (Applied Biosystems Inc., Foster City, CA).

Construction of Chimeric and Point Mutant Receptors—cDNAs of human, monkey, and rat GnRH receptors were cloned into pcDNA3.1 (+). The Tth111I unique recognition site was identified at the amino terminus/TMI junction of the human GnRH receptor. ScaI, BsGI, and StuI sites were introduced through silent mutations, as described previously, into the human and rat GnRH receptors (19). Through a series of restriction digests and or PCR amplifications using primers flanked with the appropriate restriction enzyme recognition sequences, DNA portions of both receptors were generated and gel-purified. Upon digestion of the PCR-amplified products, the receptor DNA fragments were ligated using T4 DNA ligase to assemble the rat/human chimeras. Point mutants were generated using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. Mutants are named as species receptor background, wild-type residue, residue number, and mutant residue using single letter abbreviations for the amino acids. The entire coding regions of all point mutants and chimeras were verified by automated DNA sequencing (Applied Biosystems Inc.).

Transfection—24-well tissue culture plates were seeded with the CHO-K1 cell line at a density of 150,000 cells/well in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, 50 µg/ml penicillin streptomycin, 10 mM Hepes buffer, 2 mM L-glutamine, 1 mM sodium pyruvate and incubated overnight at 37 °C, 5% CO2. After 24 h of incubation, cells were washed once with OPTI-MEM I reduced serum medium and then transfected using 3 µg of plasmid DNA to 6 µl of LipofectAMINE reagent/well. After 2 h, regular growth medium supplemented with 20% fetal bovine serum was added directly to the transfectants. Cells were incubated an additional 35–40 h at 37 °C, 5% CO2, before a radioligand competition binding assay was performed.

Binding Assay—The affinity of nonpeptide GnRH antagonists to the mutant/chimeric receptors was determined by a competitive ligand binding assay. Transfected cells were washed once with binding assay buffer (10 mM Hepes, pH 7.5, 150 mM NaCl, 0.1% bovine serum albumin, 1 mM CaCl). The assay was initiated by adding ~0.1 nM labeled [125I-Tyr5,D-Leu6,NMeLeu7,Pro9-Net]GnRH agonist and varying concentrations of competitor to the cells at 400 µl of final volume. After 1 h of incubation at 23 °C, cells were washed 4 times with cold phosphate-buffered saline and dissolved in a buffer containing 0.2 N NaOH and 1% Triton X-100. Cell lysates were transferred to polystyrene tubes, and bound radioligand was measured by direct {gamma} counting. Ki values were calculated from competition binding curves for each independent experiment using non-linear least square regression and corrected for radioligand concentration using the Cheng-Prusoff equation (20) (Prism, GraphPad Software, La Jolla, CA) assuming a KD of 0.2 nM for all mutants. Mean Ki values are calculated from the antilog of the mean of the pKi values for each receptor ligand pair.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Nonpeptide Binding To Wild-type Receptors—The affinity of three nonpeptide GnRH antagonists representing three distinct chemical families (Fig. 1) was evaluated for GnRH binding potency using a competitive radioligand binding assay. The results are shown in Table I and Fig. 2. All three compounds show high affinity binding to the human receptor (Ki = 3.4, 2.9, and 1.2 nM for compounds I, II, and III, respectively. When compounds were tested for the macaque receptor, a 25- to 40-fold decrease in affinity was observed. Even greater selectivity was seen against the rat receptor, where compound III bound with an apparent Ki of 2.4 µM and both compounds I and II bound with very low affinities (Ki >10 µM). The binding affinity of the peptide used to prepare the radioligand, [D-Leu6,NMeLeu7,Pro9-Net]GnRH, was nearly equivalent between species. Affinity of this peptide was tested for most of the mutations or chimeras discussed below, and no substantial effects on peptide affinity were observed (data not shown).



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  		FIG. 1.
Chemical structures of nonpeptide GnRH antagonists used in the present studies.

 


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  		TABLE I
Species-selective binding of nonpeptide antagonists to GnRH receptors

CHO-K1 cells were transiently transfected with GnRH wild-type receptors. Competitive binding assays as described under "Experimental Procedures" were performed to determine nonpeptide affinities. –pKi values are a mean ± S.E. from at least three independent experiments. Ki was calculated from the mean pKi value.

 



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  		FIG. 2.
Species-selective binding profile of nonpeptide antagonists to GnRH receptors. The competitive displacement of [125I-Tyr5,D-Leu6,NMeLeu7,Pro9-Net]GnRH by nonpeptide antagonists was determined using CHO-K1 cells transiently expressing wild-type human (), macaque ({circ}), or rat ({blacktriangleup}) GnRH receptors. Binding of the peptide agonist [D-Leu6,NMeLeu7,Pro9-Net]GnRH is shown for comparison. Data are mean ± S.E. of three independent experiments.

 
Residues Conferring Human/Macaque Selectivity—Comparison of human and macaque GnRH receptors revealed 8 amino acid differences, 5 of which were considered possible contacts for extracellular ligands based on locations in the extracellular or transmembrane regions (Fig. 3).3 To explore which residues might be responsible for the observed difference in nonpeptide binding affinity, we sequentially replaced each of these 5 residues in the human receptor with the macaque counterpart. As shown in Table II, mutant receptors containing point mutations at residues Ser-203 in extracellular loop II or Leu-300 in extracellular loop III showed a ~5- to 15-fold loss in nonpeptide binding affinity when replaced with the corresponding macaque residues Pro-203 or Val-300. Simultaneous introduction of both S203P and L300V mutations into the human receptor resulted in somewhat lower nonpeptide affinity than the wild-type macaque receptor for compounds I and II (240 nM compared with 87 nM for compound I and 340 compared with 120 nM for compound II). This was even more pronounced for compound III (310 nM compared with 32 nM for the macaque GnRHR). Therefore, although substitutions S203P and L300V are sufficient to account for the loss of binding affinity observed in the macaque receptor, it appears that other residues may also somewhat mitigate this loss. Conversely, replacing both residues in the macaque receptor with the corresponding human residues, macaque GnRHR(P203S,V300L), restored nearly full nonpeptide binding affinity, suggesting that these residues alone account for the observed human/macaque affinity differences.



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  		FIG. 3.
Alignment of GnRH receptor sequences from the human, rhesus macaque, and rat to bovine rhodopsin. Transmembrane helical regions are based on the crystallographically determined structure of rhodopsin (18). Locations of gaps/insertions in the alignment were adjusted to avoid regions of regular secondary structure. The alignment of extracellular loop II was based on the highly conserved Cys-196 that is disulfide-bonded to Cys-114. Numbering is based on the human GnRH receptor sequence.

 


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  		TABLE II
Residues 203 in ECL2 and 300 in ECL3 determine human/macaque selectivity for nonpeptide binding

CHO-K1 cells were transiently transfected with GnRH wild-type and mutant receptor systems. Competitive binding assays as described under "Experimental Procedures" were performed to determine nonpeptide affinities. –pKi values are a mean ± S.E. from at least three independent experiments unless noted otherwise. Ki was calculated from the mean pKi value.

 
Residues Conferring Human/Rat Selectivity—The high degree of selectivity observed between the human and rat GnRH receptors was somewhat surprising because of the high homology (87%) of these two proteins. In all, 40 residues differ between the rat and human sequences, with ~25 possibly accessible to extracellular ligands (Fig. 3).3 A chimeric approach was devised to elucidate which regions contributed to the species selectivity of nonpeptide ligands. As shown in chimeras rGnRH(h1–33), rGnRH(h1–175), and rGnRH(h1–208), nonpeptide affinity increased as the amount of human amino terminus contribution increased (Table III). For example, compound II shows no measurable binding in the rat receptor, 470 nM affinity to the rGnRH(h1–33) chimera, 270 nM affinity to the rGnRH(h1–175) chimera, and 14 nM affinity to the rGnRH(h1–208) chimera (Table III). This increase can be attributed to two specific regions. As can be seen in the rGnRH(h1–33) chimera, a greater than 10-fold increase in nonpeptide affinity is gained relative to the rGnRH wild-type by just replacing the amino-terminal domain with the corresponding human sequence. Although little or no further improvement in affinity is observed by the addition of the human 34–175 region (comparing the rGnRH(h1–33) and rGnRH(h1–175) chimeras), a 30- to 40-fold increase in affinity, depending on compound, is gained in the rGnRH(h1–208) relative to the rGnRHh(h1–33) chimera, indicating that additional residues in extracellular loop II are contributing substantially to species selectivity. This was confirmed in the rGnRH(h1–33, 176–208) chimera where nonpeptide affinity for compound I is 80 nM, compound II is 42 nM, and compound III is 23 nM (Table III). Although these two regions restore most of the affinity when present in combination, it should be noted that the chimera containing extracellular loop II by itself failed to gain sufficient affinity to allow measurement of binding affinity.


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  		TABLE III
Rat/human chimeras localize nonpeptide selectivity determinants to the amino terminus and ECL2

Chimeras were constructed by replacing regions of the rat GnRH receptor with comparable regions of the human GnRH receptor. Competitive binding assays as described under "Experimental Procedures" were performed to determine nonpeptide affinities. –pKi values are a mean ± S.E. from at least three independent experiments. Ki was calculated from the mean pKi value.

 
To further map residues responsible for selectivity in the amino terminus and extracellular loop II regions, two point mutation studies were carried out. As shown in Table IV, 5 residues within the rat amino terminus, Asn-4, Leu-7, Asp-10, Thr-24, and Lys-27, were systematically mutated to the human counterpart. Of the five point mutants, only the T24M receptor showed a measurable gain in affinity. Compound I bound to the rGnRHR(T24M) point mutant with an apparent Ki of 4.3 µM, compound II bound with an apparent Ki of 3.5 µM, and compound III bound with an apparent Ki of 500 nM (Table IV). The reciprocal mutation, hGnRHR(M24T), resulted in reduction of nonpeptide affinity for the human receptor by approximately three orders of magnitude, suggesting that Met-24 is critical for high affinity binding to the human receptor. However, although this point mutation reduced affinity of the human receptor for compound III to that of the rat receptor, compounds I and II still bound with higher affinity than the rat receptor, suggesting that the binding mode of compound III is somehow different from I and II. Simply removing the threonine hydroxyl by replacing Thr-24 with alanine in the rat receptor did not improve binding, suggesting that reduced affinity is not a disrupting effect of the threonine residue. Although emphasis can be placed on the 24th position, it is evident that no single point mutant restores binding affinity to the same degree as seen in the rGnRH(h1–33) chimera, indicating that combinations of more subtle interactions between Met-24 and other residues in the region may also contribute to nonpeptide affinity, albeit to a lesser degree. Further, if one compares the nearly 5-fold improvement in binding of compound III achieved by the rGnRHR(T24M) mutant relative to the native rat receptor with the nearly 2000-fold loss in affinity caused by the reciprocal hGnRHR(M24T) mutation in the human receptor, it appears likely that Met-24 is involved in organizing an extracellular region of the receptor for high affinity nonpeptide binding, rather than simply contributing a direct interaction with the ligand.


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  		TABLE IV
Amino-terminal residue Met-24 is required for high affinity binding of nonpeptides to the human GnRH receptor

CHO-K1 cells were transiently transfected with GnRH wild-type, chimeric, and mutant receptor systems. Competitive binding assays as described under "Experimental Procedures" were performed to determine nonpeptide affinities. –pKi values are a mean ± S.E. from at least three independent experiments. Ki was calculated from the mean pKi value.

 
To localize residues in extracellular loop II that confer the binding activity of the 176–208 region of the human receptor, the 7 residues that differ between rat and human in this region (Tyr-182, Gly-186, Pro-189, Ala-190, {Delta}-191, Pro-203, and Glu-208) were systematically mutated to the human counterpart. To obtain measurable changes in binding affinity, these point mutants were constructed within the context of the r-GnRH(h1–33) chimera. As shown in Table V, the rGnRHR(h1–33,E208Q) mutation showed the largest improvement in affinity for all three compounds and E208Q could account for the bulk of binding energy provided for by the human 176–208 region. Compound I bound to the E208Q mutant with an apparent Ki of 110 nM, compound II bound with an apparent Ki of 83 nM, and compound III bound with an apparent Ki of 52 nM, as compared with 80, 42, and 23 nM, respectively, for the complete human ECL2 region. Replacement of this residue alone, without the human 1–33 sequence (rGnRHR(E208Q)), did not observably improve binding. However, changing this residue alone in the human sequence to the rat side chain (hGnRHR(Q208E)) resulted in a 10- to 25-fold loss in potency. It therefore appears that although Gln-208 is necessary for high affinity nonpeptide binding, like Met-24 this residue is required for organizing an extracellular region but cannot alone substantially interact with nonpeptide ligands. A smaller contribution of the P203S to nonpeptide binding was also observed, consistent with the observations in the macaque receptor.


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  		TABLE V
Gln-208 at the ECL2/TM5 interface is required for high affinity binding of nonpeptides to the human GnRH receptor

CHO-K1 cells were transiently transfected with GnRH wild-type, chimeric, and mutant receptor systems. Competitive binding assays as described under "Experimental Procedures" were performed to determine nonpeptide affinities. –pKi values are a mean ± S.E. from at least three independent experiments. Ki was calculated from the mean pKi value.

 
Although replacement of the amino terminus and ECL2 in the rat receptor by the corresponding human sequences in the rGnRH(h1–33, 176–208) chimera resulted in substantial improvement in nonpeptide binding affinity, the full affinity of the human receptor was not fully restored, indicating that additional residues are required. Both rat and macaque GnRH receptors differ from the human receptor by the presence of Val-300 (Leu-300 in the human). As was discussed above, the hGnRH(L300V) point mutant decreases nonpeptide affinity by 9- to 14-fold. When the reciprocal mutation (rGnRHR(V300L)) was introduced into the rat receptor, a measurable affinity was regained for nonpeptides I and II relative to the wild-type rGnRHR (Table VI); however, no effect on binding of compound III was observed, again suggesting a somewhat different mode of binding for compound III. To determine whether the effect of this residue was additive or cooperative with other regions, the V300L substitution was introduced into the rGnRH(h1–33, 176–208) chimera. The resulting chimera, rGnRH(h1–33, 176–208,V300L), binds compound I with an apparent Ki of 7.3 nM, compound II binds with an apparent Ki of 2.7 nM, and compound III binds with an apparent Ki of 1.4 nM, corresponding to an 11- to 16-fold improvement in affinity and essentially restoring full human receptor affinity. However, when the V300L substitution is introduced with T24M alone, or in the rGnRH(h176–208/T24M) construct, only a 5- to 8-fold increase in affinities of all three nonpeptides is observed, suggesting that the full contribution of Leu-300 requires amino-terminal residues in addition to Met-24. Further evidence for cooperative action with other residues in the amino terminus and ECL2 comes from introduction of all four major humanizing mutations into the rat receptor (rGnRH(T24M,P203S,E208Q,V300L)), which still has 70-, 50-, and 20-fold reduced affinity for compounds I, II and III, respectively, compared with the native human receptor.


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  		TABLE VI
Ability of extracellular residues and regions to restore high affinity nonpeptide binding to the rat GnRH receptor

CHO-K1 cells were transiently transfected with GnRH wild-type, chimeric, and mutant receptor systems. Competitive binding assays as described under "Experimental Procedures" were performed to determine nonpeptide affinities. –pKi values are a mean ± S.E. from at least three independent experiments unless noted otherwise. Ki was calculated from the mean pKi value.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
A range of nonpeptide chemical entities that act as antagonists at the human GnRH receptor have been described (7) and offer significant potential for novel therapeutics to treat diseases related to the reproductive endocrine axis. However, little is known about how these molecules interact with the GnRH receptor. To investigate residues in the receptor that are important for nonpeptide binding, we took advantage of the differences in affinity for the human and non-human receptors that have been observed for many of these compounds. Chimeric receptors and mutations of individual residues were employed to evaluate effects on binding of representatives of three classes of nonpeptide antagonists.

The results presented above demonstrate that residues in the amino terminus and extracellular loops II and III of the GnRH receptor account for the species-selective binding observed for three classes of nonpeptide antagonists. Four key residues appear to be primarily responsible, Met-24, Ser-203, Gln-208, and Leu-300. Met-24, located in the amino-terminal extracellular domain, was clearly the most critical residue for high affinity binding to the human receptor. Replacement of Met-24 with the corresponding rat residue (hGnRHR(M24T)) resulted in an approximately three orders of magnitude loss of affinity for all three nonpeptide ligands (Table IV). The replacement of human residues in extracellular loops II and III with the corresponding rat/macaque residues in the hGnRHR(S203P) and hGnRHR(L300V) mutants had pronounced, but lesser, effects on affinity of all three compounds (5- to 14-fold) (Table II), whereas the hGnRHR(Q208E) mutant at the junction of TM5 and ECL-II had 7- to 27-fold reductions in binding affinity (Table V).

Interestingly, all 4 residues are located in the putative extracellular portions of the receptor (Fig. 4). This is in contrast to the observations of Cui et al. (9), who showed that Phe-313 is critical for human/dog species-selective binding (60- to 330-fold) of quinolone-based nonpeptide GnRH antagonists. This residue is in the middle of TM7 and homologous to Lys-296, which is linked to retinal in rhodopsins. Further, these authors note that Ser-203 and Leu-300 were not involved in determining species selectivity for the quinolone-based nonpeptide GnRH antagonists, suggesting that quinolones and the three classes of nonpeptide shown here utilize distinct receptor interactions for binding. This finding of selectivity determinants in the amino terminus and extracellular loops is also in contrast to most other studies, which have typically localized nonpeptide contact residues in the transmembrane regions of peptide-activated class A G protein-coupled receptors, including the cholecystokinin receptor, CCK1R (21), neuromedin B receptor (22), angiotensin AT1 (23), growth hormone secretagogue receptor, GHS-R (24), neurotensin receptor NTR1 (25), and tachykinin receptor, NK2 (26).



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  		FIG. 4.
Schematic representation of the human GnRH receptor based on alignment to bovine rhodopsin shown in Fig. 3. Transmembrane helical regions are based on the crystallographically determined structure of rhodopsin (18). Disulfide bonds are based on cross-linking and mutagenesis studies previously described (28). Locations of residues involved in determining nonpeptide species selectivity are shown. Numbering is based on the human GnRH receptor sequence.

 
However, involvement of the extracellular domains for nonpeptide binding is not unprecedented. One example similar to the present finding is described for nonpeptide binding to the human V1 vasopressin receptor: the E324D mutation in ECL-III shows some improvement in affinity for the nonpeptide OPC21268 but when this mutation is combined with G337A in TM-VII a dramatic cooperativity is observed (27). Because 2 additional residues in the transmembrane helices were linked to nonpeptide affinity in this receptor, it is likely that the nonpeptide binding site for the V1 receptor is formed by a combination of TM and ECL-III residues. Further, roles for residues in the amino terminus and extracellular loops II and III have previously been described for GnRH peptides. Photoreactive GnRH peptide agonist (28) and antagonist (29) have both been shown to react with Cys-14, indicating that peptide ligand binding may also involve the amino-terminal region of the receptor. Mutations in extracellular loop II have been shown to convert peptide antagonists to agonists (19), and Asp-302 in extracellular loop III has been shown to interact with the basic Arg-8 in mammalian GnRH peptides (30).

Although the effects of various mutations are generally correlated between the three nonpeptides studied here, not surprisingly some quantitative differences in the effects of mutations are observed. For example, the hGnRHR(S203P,L300V) construct has 70-fold lower affinity for compound I compared with the human receptor but 260-fold reduced affinity for compound III, resulting in equivalent potency for the three compounds. Similarly, hGnRHR(M24T) reduces affinity for all three compounds but eliminates potency differences seen in the wild-type human receptor. The effect of the Q208E mutation in the human receptor results in a 27-fold reduction in affinity for compound I but only 7-fold for compound III. Residues other than Met-24 in the amino terminus appear to be more important for binding of compound II than compound I, based on the reduced ability of T24M to restore affinity compared with the human 1–33 sequence in the context of the human ECL2 and V300L substitutions (comparing rGnRHR(h176–208,T24M,V300L) with rGnRHR(h1–33,176–208,V300L) in Table VI). Finally, the quadruple mutant, rGnRH (T24M,P203S,E208Q,V300L) is more effective at restoring affinity for compound III than for compound I (Table VI). Together, these observations suggest that somewhat different receptor interactions are utilized by the three nonpeptide ligands, although the exact molecular nature of these differences remains to be elucidated.

Comparison of the ability of various substitutions to disrupt binding in the human receptor with the ability of the reciprocal mutations to confer high affinity binding to the rat receptor appears to suggest that they may be involved in organizing a receptor region required for high affinity nonpeptide binding rather than providing direct interactions with these ligands. For example, the T24M substitution provides only modest improvement for the rat receptor, but the reciprocal M24T mutation in the human receptor results in approximately three orders of magnitude loss of affinity. Similarly, the E208Q substitution provides no measurable improvement in the rat receptor, but the reciprocal Q208E mutation in the human receptor causes a 7- to 27-fold reduction in affinity. However, a 3- to 8-fold improvement in affinity can be observed for the E208Q substitution when the human 1–33 region is also present. Similarly, the entire human ECL2 region had no observable improvement in affinity when introduced into the rat receptor, but when introduced together with the human 1–33 region a significant improvement was observed. These data suggest that the amino terminus, ECL2, and ECL3 coordinately contribute to high affinity binding of these classes of nonpeptide ligands.

In summary, we have shown that residues in the amino terminus and extracellular Loops II and III of the GnRH receptor are responsible for high affinity binding of three classes of nonpeptide GnRH antagonists. Comparisons of a collection of reciprocal mutations suggest that these may not be direct contacts but rather appear to be involved in organizing extracellular portions of the receptor into a three-dimensional structure that is able to bind these compounds with high affinity. The overall binding modes of the three nonpeptides presented here appear to be similar but not identical and are distinct from a fourth class of GnRH nonpeptide antagonist reported to be dependent on a residue more deeply located in the transmembrane region. These data may be useful in reconciling structure activity relationships of various chemical series and provide useful constraints for molecular modeling of GnRH receptor ligand interactions.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grants 1-R43-HD38625-01 and 2-R44-HD38625-02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** To whom correspondence should be addressed: Neurocrine Biosciences Inc., 10555 Science Center Dr., San Diego, CA 92121. Tel.: 858-658-7740; Fax: 858-658-7601; E-mail: sstruthers{at}neurocrine.com.

1 The abbreviations used are: GnRH, gonadotropin-releasing hormone; hGnRHR-R, human GnRHR; GnRHR, gonadotropin-releasing hormone receptor; rGnRH-R, rat GnRHR; TM, transmembrane domain; CHO, Chinse hamster ovary; ECL, extracellular loop. Back

2 F. C. Tucci, Y-F. Zhu, Z. Guo, T. D. Gross, J. Saunders, P. J. Connors, Jr., M. W. Rowbottom, Y. Gao, R. S. Struthers, Q. Xie, G. J. Reinhart, P. J. Conlon, A. K. L. Bonneville, T. K. Chen, H. Bozigian, N. Ling, X.-J. Liu, and C. Chen, manuscript in preparation. Back

3 The rat receptor also differs from the human by a single amino acid deletion corresponding to Lys-191 in the human receptor. To simplify the discussion, the corresponding human residue numbers are used to refer to amino acid positions in the rat sequence. Thus, residues 191–327 in the actual rat sequences are referred to as 192–328 to account for the deletion of Lys-191. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Robert Millar, Sam Hoare, and Tom Ott for helpful advice and discussions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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