Identification of Phe313 of the Gonadotropin-Releasing Hormone (GnRH) Receptor as a Site Critical for the Binding of Nonpeptide GnRH Antagonists

Jisong Cui, Roy G. Smith, George R. Mount, Jane-L. Lo, Jinghua Yu, Thomas F. Walsh, Suresh B. Singh, Robert J. DeVita, Mark T. Goulet, James M. Schaeffer and Kang Cheng

Department of Endocrinology and Chemical Biology (J.C., J-L.L., J.Y., J.M.S., K.C.) Department of Medicinal Chemistry (T.F.W., R.J.D., M.T.G.) Department of Molecular Systems (S.B.S.) Merck Research Laboratories Rahway, New Jersey 07065
Huffington Center of Aging (R.G.S.) Houston, Texas 77030
Temple University (G.M.) School of Medicine Philadelphia, Pennsylvania 19140


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The dog GnRH receptor was cloned to facilitate the identification and characterization of selective nonpeptide GnRH antagonists. The dog receptor is 92% identical to the human GnRH receptor. Despite such high conservation, the quinolone-based nonpeptide GnRH antagonists were clearly differentiated by each receptor species. By contrast, peptide antagonist binding and functional activity were not differentiated by the two receptors. The basis of the differences was investigated by preparing chimeric receptors followed by site-directed mutagenesis. Remarkably, a single substitution of Phe313 to Leu313 in the dog receptor explained the major differences in binding affinities and functional activities. The single amino acid replacement of Phe313 of the human receptor with Leu313 resulted in a 160-fold decrease of binding affinity of the nonpeptide antagonist compound 1. Conversely, the replacement of Leu313 of the dog receptor with Phe313 resulted in a 360-fold increase of affinity for this compound. These results show that Phe313 of the GnRH receptor is critical for the binding of this structural class of GnRH antagonists and that the dog receptor can be "humanized" by substituting Leu for Phe. This study provides the first identification of a critical residue in the binding pocket occupied by nonpeptide GnRH antagonists and reinforces cautious extrapolation of ligand activity across highly conserved receptors


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GnRH is a decapeptide synthesized in the medial basal hypothalamus and released in a pulsatile manner into the hypophyseal portal circulation (1). GnRH binds with high affinity to its receptors on gonadotrope membranes of the anterior pituitary resulting in the release of LH and FSH. The GnRH-receptor interaction activates phospholipase C ß-isoforms via the G protein, Gq/G11, resulting in an increased phospholipid turnover and the formation of inositol 1,4,5-trisphosphate and diacylglycerol. The ensuing increase in cytoplasmic calcium and activation of protein kinase C leads to the synthesis and secretion of gonadotropins. LH released from the pituitary gland is primarily responsible for the regulation of gonadal steroid production in both sexes, whereas FSH regulates spermatogenesis in males and follicular development in females (2).

The GnRH receptor was first cloned from mouse in 1992 (3), and homologous receptors were soon identified in human and several other mammalian species (4, 5, 6, 7, 8, 9). This receptor belongs to the superfamily of G protein-coupled receptors (GPCRs) with seven transmembrane (TM) domains. The GnRH receptor has more than 80% overall identity in mammals and is highly conserved within the putative TM domains. A unique feature of the mammalian GnRH receptors is the absence of a cytoplasmic carboxyl-terminal tail which has been implicated in rapid receptor desensitization (2).

GnRH peptide agonists have been widely used for the treatment of prostate cancer; however, the agonists, as expected, stimulate gonadotropin release, resulting in increased secretion of gonadal steroids to produce a flare response. However, after 1–2 weeks of chronic treatment, the receptor desensitizes and the compounds act as functional antagonists leading to suppression of secretion of gonadotropins and gonadal steroids. By contrast, antagonists have rapid onset of inhibitory action, hence avoiding the flare (10, 11, 12, 13). Dogs make ideal subjects for evaluating functional efficacy in vivo. Therefore, we cloned and characterized the dog GnRH receptor to allow evaluation of the potency of antagonists of this receptor and to make comparisons with potency on the human GnRH receptor. The dog GnRH receptor has marked decreased affinity in the binding of the quinolone-based nonpeptide antagonists but retains a similar affinity for peptide GnRH ligands. The basis of these differences was elucidated by site-directed mutagenesis.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cloning and Characterization of the Dog GnRH Receptor
Attempts to directly clone the full-length coding cDNA of the dog GnRH receptor by RT-PCR from dog pituitary mRNA using degenerate primers derived from the conserved N- and C-terminal amino acids of the mammalian GnRH receptors were unsuccessful. This suggests that the 5'- and/or 3'-sequences of the dog GnRH receptor are different from other mammalian GnRH receptors. Partial cDNAs were cloned from the dog genomic GnRH receptor by PCR using degenerate primers derived from the amino acids of the conserved TM domains. The 5' and 3' specific coding sequences of the dog GnRH receptor were determined from genomic clones, and the full-length coding cDNA was cloned from the pituitary mRNA by RT-PCR using specific primers described in Materials and Methods. Sequence analysis revealed that the dog GnRH receptor contained 327 amino acids1 (Fig. 1Go). The dog receptor has 92.6% identity and 96.6% similarity to the human receptor. In addition, it has well conserved TM domains and the sites for glycosylation and for disulfide bond formation. The dog receptor also lacks a C-terminal cytoplasmic tail (Fig. 1Go), a common characteristic of all mammalian GnRH receptors. The sequence of the dog receptor suggested that it was one residue shorter than the human receptor by lacking the amino acid residue Asn3 present in the human receptor. This particular residue is conserved in all other known GnRH receptors of mammalian species (2), and absence of this conserved residue near the N terminus may explain why our approach using degenerate primers based on the N- and C-terminal sequences was unsuccessful.



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Figure 1. Sequence Comparison between the Human (h-R) and Dog (d-R) GnRH Receptors

The amino acid sequence is shown in single letter and numbered at right. The putative transmembrane domains 1–7 are indicated by Roman numbers I-VII on the top. The amino acid differences between the two receptors are in bold. Putative glycosylation sites are in italics, and the cysteine residues for disulfide bond formation are underlined. Phe313 of the human receptor (Leu312 of the dog receptor) is indicated by a star.

 
It was possible that the missing amino acid in the dog receptor was a rare event that occurred in the dog genomic library from a single donor. To investigate this possibility, pituitary mRNA was individually isolated from six dogs, and RT-PCR was performed to clone the cDNA spanning the first 200 bp of the open reading frame. All six dogs lacked the same asparagine residue (data not shown).

In Vitro Evaluation of Nonpeptide GnRH Antagonists on the Dog GnRH Receptor
A series of structurally related nonpeptide quinolone-based GnRH antagonists (Refs. 14, 15, 16 and R. J. Devita, M. Parikh, J. Jiang, M. T. Goulet, M. J. Wyvratt, J-L. Lo, Y. T. Yang, J. Cui, N. Ren, K. Cheng, and R. G., Smith, manuscript in preparation) were evaluated for inhibition of GnRH-stimulated inositol phosphate (IP) production in CHO-K1 cells transiently expressing either dog or human GnRH receptors. Treatment with all compounds in cells expressing the human GnRH receptor decreased IP production in a dose-dependent manner with IC50 values ranging from 0.8 to 33.8 nM (Table 1Go). However, the IC50 values determined using the cells expressing the dog receptor were approximately 50- to 300-fold higher (Table 1Go). Figure 2AGo shows the inhibition of GnRH-stimulated IP production in the presence of various concentrations of compound 1 (16) in cells expressing either dog or human GnRH receptors. Compound 1 was used in subsequent characterization of the mutant human and dog GnRH receptors.


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Table 1. The Structure and Potency of Nonpeptide GnRH Antagonists on the Human and Dog GnRH Receptors in the PI Turnover Assay

 


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Figure 2. Characterization of the Dog and Human GnRH Receptors

CHO-K1 cells were transiently transfected with pcDNA3.1 carrying cDNA encoding human (•) or dog ({blacksquare}) GnRH receptors. A, Transfected cells were incubated with 3H-inositol phosphates for 24 h at 37 C. After the incubation, cells were treated with various concentrations of compound 1 for 2 h before the addition of 1 nM GnRH. After incubation at 37 C for 1 h, cells were lysed and the cell extract was collected and countered. The inhibition of GnRH-stimulated IP production by compound 1 was in a dose-dependent manner. B, Transfected cells were incubated with 0.1 nM 125I-buserelin at 22 C for 1 h in the presence of various concentrations of compound 1. After the incubation, cells were dissolved and the cell suspension was collected and counted. The inhibition of specific 125I-buserelin binding by compound 1 was in a dose-dependent manner. The X- and Y-axis indicates the concentration of compound 1 (nM) and the percentage of inhibition, respectively, and the IC50 values (nM) are shown in the upper left corner (also for Figs. 3Go, 4Go, and 6Go).

 
Compound 1 was evaluated for its binding affinity to human or dog GnRH receptors transiently expressed in CHO-K1 cells. Consistent with PI turnover assays, compound 1 binds to the dog receptor with 364-fold less affinity than to the human receptor (IC50 value of 1420 vs. 3.9 nM) (Fig. 2BGo), suggesting that the dog receptor is missing some critical sites for binding this nonpeptide GnRH analog.

The binding affinity of a series of peptide GnRH ligands was also determined on the dog receptor. These peptide analogs bind to the dog and human GnRH receptors with a similar affinity (Table 2Go), indicating that the dog receptor does contain the critical sites for binding these peptide ligands. Figure 3Go shows the binding of 125I-buserelin (17) to the dog and human GnRH receptors in the presence of the peptide antagonist cetrorelix (12) in the whole-cell binding assay. Cetrorelix has an IC50 value of 0.52 and 0.55 nM on the dog and human receptors, respectively.


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Table 2. The Potency of Peptide GnRH Ligands on the Human and Dog GnRH Receptors in the Whole-Cell Binding Assay

 


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Figure 3. Inhibition of Specific 125I-Buserelin Binding by Cetrorelix

CHO-K1 cells were transiently transfected with pcDNA3.1 carrying the cDNA encoding human (•) or dog ({square}) GnRH receptors. Transfected cells were incubated with 125I-buserelin at 22 C for 1 h in the presence of various concentrations of cetrorelix. After the incubation, cells were dissolved and the cell suspension was collected and counted. The inhibition of specific 125I-buserelin binding by cetrorelix was in a dose-dependent manner.

 
The C Terminus of the Dog GnRH Receptor Contains Amino Acid Residues That Discriminate the Quinolone-Based Nonpeptide GnRH Antagonist from Peptide Ligands
To identify the amino acid residues critical for the binding of compound 1, two chimeric receptor proteins were constructed between the human and dog GnRH receptors using two restriction sites (HindIII and PstI) commonly occurring in both receptors (Fig. 4AGo). The first chimera (chi-1) consists of one-third N terminus of the dog and two thirds C terminus of the human receptor, and the second chimera (chi-2) contains two thirds N terminus of the dog and one third C terminus of the human receptor (Fig. 4AGo). Compound 1 binds with high affinity to both chi-1 and chi-2 with an IC50 value of 2.4 and 3.1 nM, respectively. These IC50 values are similar to the IC50 of 3.9 nM on the human receptor (Fig. 4BGo). Compound 1 had an IC50 of 11.3 and 14.0 nM in the PI turnover assay on the two chimeras that was even lower than that of the human receptor (33.8 nM) (Fig. 4CGo). These results suggest that the C terminus of the dog receptor contains residues that form a binding pocket capable of discriminating between different structural classes of antagonists.



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Figure 4. Biological Activities of the Dog-Human Chimeric GnRH Receptors

A, Schematic presentation of the chimeric receptors. The dog receptor is shown as a striped bar and the human sequence as an open bar. The restriction sites relative to the amino acid sequence of the human GnRH receptor are indicated by the numbers in parentheses. B, CHO-K1 cells were transiently transfected with pcDNA3.1 carrying cDNA encoding chi-1 ({square}), chi-2 ({diamondsuit}), human (•), or dog ({blacksquare}) GnRH receptors. Transfected cells were incubated with 125I-buserelin at 22 C for 1 h in the presence of various concentrations of compound 1. After the incubation, cells were dissolved and the cell suspension was collected and counted. The inhibition of specific 125I-buserelin binding by compound 1 was in a dose-dependent manner. C, Transfected CHO-K1 cells were incubated with 3H-inositol phosphates for 24 h at 37 C. After the incubation, cells were treated with various concentrations of compound 1 for 2 h before the addition of 1 nM GnRH. After incubation at 37 C for 1 h, cells were lysed and the cell extract was collected and countered. The inhibition of GnRH-stimulated IP production by compound 1 was in a dose-dependent manner.

 
Identification of Phe313 as a Major Binding Site for This Class of Nonpeptide Antagonist
The C-terminal fragment of the human and dog GnRH receptors (residues 201 to 328) differs at five residues. Sequence comparison at these five positions among GnRH receptors from human, monkey, dog, and rat is summarized in Fig. 5Go. In a separate study, it was demonstrated that compound 1 binds with a similar affinity to both monkey and human GnRH receptors (J. Cui, J-L. Lo, G. R. Mount, and K. Cheng, unpublished data), suggesting that the residues at positions 203 and 300 were not the determinants. Although it was less potent in binding to the rat receptor than to the human receptor, compound 1 still has a 20-fold higher affinity against the rat receptor than the dog receptor (data not shown). This result implies that Lys264 is also not a critical determinant for the binding of compound 1. Thus, the two remaining residues of the dog receptor, Thr227 and Leu313, should contain the amino acid(s) that distinquish this class of nonpeptide GnRH ligands.



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Figure 5. Amino Acid Comparison at Positions 203, 227, 264, 300, and 313 of the GnRH Receptors of Human, Monkey, Dog, and Rat

The position is indicated by the number in italics at top, according to the human receptor. Residues unique to the dog receptor are underlined.

 
Thr227 or Leu313 or both were introduced into the human receptor (h-R227T, h-R313L, and h-R227T, 313L) by site-directed mutagenesis. Conversely, the corresponding mutations were also introduced into the dog receptor (d-R227 M, d-R313F, and d-R227 M, 313F). In cells expressing h-R227T, 313L, d-R227 M, 313F, and d-R227 M, no receptor binding or functional response was detected using the peptide ligands such as GnRH, buserelin, and cetrorelix (data not shown), indicating that the mutant receptors were either not expressed or expressed in an inappropriate form. The single mutant h-R227T was indistinguishable from the wild-type human receptor in both binding and functional assays (Fig. 6Go, A and B), indicating that Met227 of the human receptor is not a critical determinant for binding this nonpeptide ligand. In contrast, the mutation at position 313 (h-R313L and d-R313F) resulted in a significant change in receptor affinity toward this nonpeptide ligand. The replacement of Phe313 of the human receptor with Leu313 resulted in a substantial decrease in the binding affinity for compound 1. Compound 1 had an IC50 of 627 nM in the binding assay and 955 nM in PI turnover assay on the mutant human receptor h-R313L. These values are comparable to that of the wild-type dog receptor (Fig. 6Go, A and B). Similarly, the mutant dog receptor d-R313F displayed a 414-fold increase in binding affinity for compound 1 compared with the wild-type dog receptor with an IC50 of 3.5 nM, which was similar to that of the wild-type human receptor (3.9 nM) (Fig. 6AGo). In the PI turnover assay, d-R313F gained 48-fold activity in response to Compound 1 with an IC50 of 40 nM. These results demonstrate that Phe313 of the human receptor is critical for the binding of the nonpeptide GnRH antagonist compound 1.



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Figure 6. Characterization of the Mutant GnRH Receptors

CHO-K1 cells transiently transfected with pcDNA3.1 carrying the cDNA encoding human (•), dog ({blacksquare}), h-R227T ({circ}), h-R313L ({square}), or d-R313F ({triangleup}) GnRH receptors were used in the whole cell binding and PI turnover assays. A, transfected cells were incubated with 125I-buserelin at 22 C for 1 h in the presence of various concentrations of compound 1. After the incubation, cells were dissolved and the cell suspension was collected and counted. The inhibition of specific 125I-buserelin binding by compound 1 was in a dose-dependent manner. B, Transfected cells were incubated with 3H-inositol phosphates for 24 h at 37 C. After the incubation, cells were treated with various concentrations of compound 1 for 2 h before the addition of 1 nM GnRH. After incubation at 37 C for 1 h, cells were lysed and the cell extract was collected and countered. The inhibition of GnRH-stimulated IP production by compound 1 was in a dose-dependent manner. C, Transfected cells were incubated with 125I-buserelin at 22 C for 1 h in the presence of various concentrations of cetrorelix. After the incubation, cells were dissolved and the cell suspension was collected and counted. The inhibition of specific 125I-buserelin binding by cetrorelix was in a dose-dependent manner.

 
The binding of peptide antagonist cetrorelix to the mutant receptors was also evaluated in the whole-cell binding assay. Cetrorelix binds to all three single mutant receptors (h-R227T, h-R313L, and d-R313F) and the wild-type human and dog receptors (h-RWT and d-RWT) with a comparable affinity (Fig. 6CGo). In addition, these mutants are indistinguishable from the wild-type human and dog receptors in mediating the GnRH-stimulated IP production (data not shown). These data suggest that Phe313 is not critical for the binding of peptide ligands.

The maximal binding (Bmax) of the wild-type and mutant GnRH receptors was calculated from the competition binding assay (Fig. 6CGo) to determine the expression levels of these receptors. The wild-type dog receptor (d-RWT) and its mutant at 313 (d-R313F) have a Bmax of 16,460 and 25,320 cpm/well, respectively, while the wild-type human receptor (h-RWT) and the mutant h-R313L have a maximal binding of 7,430 and 8,472 cpm/well. The expression of mutant h-R227T was slightly lower than others with a Bmax of 5,059 cpm/well; however, this mutant has an equivalent binding affinity for both peptide and nonpeptide ligands as the wild-type human receptor (Fig. 6Go). These data indicate that the receptor expression of the two critical mutants, h-R313L and d-R313F, is comparable to their wild-type parent receptors.

The Quinolone Ring of Compound 1 Interacts with the Side Chain of Phe313 of the GnRH Receptor
Computer models of compound 1 docked into the human and dog GnRH receptors are shown in Fig. 7Go, A and B. The side chains of Phe313 or Leu313 in these models are facing the quinolone ring of compound 1. The difference in the surface area between these two side chains interacting with the quinolone moiety is approximately 90 Å2, which contributes approximately 2.25 kcal/mol (0.025 kcal/Å2). This is equivalent to an 100-fold decrease in binding affinity of compound 1 ({Delta}G = -RTln(100) = 2.74 kcal/mol) to the dog GnRH receptor when compared with that of the human receptor.



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Figure 7. A Model of Compound 1 Docked into the Human (A) and Dog (B) GnRH Receptors

The TM domains of the human and dog GnRH receptors (see Fig. 1Go) were aligned with a ß2-adrenergic receptor model (26 ) using the homology modeling software LOOK V3.5 (Molecular Applications Group, Palo Alto, CA) with the manual alignment based on the automated homology modeling and energy minimization procedures SEGMOD (27 ) and ENCAD (28 ), respectively, implemented in LOOK. The color is coded according to the residue type: red for acidic residues (Asp and Glu); blue for basic residues (Arg and Lys); green for polar residues (Asn, Gln, His, Ser, and Thr); yellow for Cys; white for nonpolar residues (Ala, Gly, Ile, Leu, Met, Pro, and Val); and gray for aromatic residues (Phe, Tyr, and Trp). The compound 1 carbon skeleton is colored in yellow, oxygens in red, and nitrogens in blue, and the amino acid residues Asp302, Phe313 (or Leu313), and Lys121 in white.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The primary finding of this study was the cloning and characterization of the dog GnRH receptor and the identification of Phe313 of the GnRH receptor as an important component in the formation of the major binding pocket for quinolone-based nonpeptide GnRH antagonists. Although the structure and function of the dog GnRH receptor are substantially similar to the GnRH receptors of other mammals, it is unique in several aspects. First, the dog receptor lacks the residue Asn3 present in the human receptor. Although it is conserved in all other known mammalian GnRH receptors, this residue appears not to be essential for ligand binding and functional response since the mutant dog receptor d-R313F and the two chimeric receptors without this residue had IC50 values similar to the wild-type human receptor for both peptide and nonpeptide ligands that were evaluated in the whole-cell binding assay and PI turnover assay. Second, the dog GnRH receptor lacks the major binding site for the quinolone-based nonpeptide GnRH antagonists. The single substitution of Phe313 with Leu313 in the dog receptor results in a significant decrease in the binding affinity for this class of nonpeptide ligands. The importance of Phe313 for the binding of compound 1 was demonstrated by the site-directed mutagenesis study. The single change of Phe313 of the human receptor to Leu313 resulted in an 160-fold decrease in the binding affinity of the nonpeptide antagonist compound 1, while the dog receptor with the replacement of Leu313 with Phe313 gains more than 400-fold binding affinity for this ligand.

Phe313 is conserved in all other known mammalian GnRH receptors. However, this residue has not previously been demonstrated to be critical for the binding of GnRH and its synthetic peptide ligands. Our studies also show that alteration at position 313 of the GnRH receptor did not change the binding affinity for peptide ligands. These results suggest that the critical binding sites for the quinolone-based nonpeptide ligands do not completely overlap with that for the peptide ligands.

Computer modeling (Fig. 7Go, A and B) suggests a potential binding pocket for compound 1 in the GnRH receptor. Consistent with the results of site-directed mutagenesis, this model predicts that the quinolone ring of compound 1 interacts with the side-chain of Phe313. This model also predicts that the residues of Lys121 and Asp302 are critical for the binding of compound 1. The basic nitrogen of the piperidine ring of compound 1 is 3.1Å from the nearest carboxylate oxygen of Asp302, and the carbonyl oxygen of this compound is at a distance of 2.9 Å from the {epsilon}NH2 of Lys121 (Fig. 8Go). This model suggests that the side chain of residues of Phe313, Lys121, and Asp302 form a binding pocket for compound 1. The residues of Asp98, Asn102, Lys121, and Asp302 were previously identified as the major binding sites for GnRH and its peptide analogs (18, 19, 20, 21). Thus, Lys121 and Asp302 may be involved in the binding of both nonpeptide and peptide ligands. If this is the case, it suggests that the binding pocket for these two types of GnRH ligands partially overlap. Consistent with this hypothesis, the non-peptide ligand compound 1 competitively displaces the binding of 125I-labeled peptide ligand buserelin (Fig. 2BGo).



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Figure 8. Schematic Presentation of the Distance from the Basic Nitrogen of the Piperidine Ring and the Carbonyl Oxygen of Compound 1 to Asp302 and Lys121, Respectively, of the GnRH Receptor in the Model Shown in Fig. 7Go

The residues Phe313, Asp302, and Lys121 are in bold. The quinolone ring of compound 1 is interacting with the side chain of Phe313. The distance of the piperidine ring to Asp302 is 3.1 Å, and the distance of the carbonyl oxygen to Lys121 is 2.9 Å.

 
There are other examples that the single amino acid alteration leads to significant change in receptor functionality. Deletion of Lys191 from the human GnRH receptor caused a 4-fold increase in receptor expression (22). Zhou et al. (19) reported that Lys121 of the GnRH receptor differentiates agonist and antagonist binding sites.

Nonpeptide GnRH antagonists structurally distinct from Compound 1 have also been investigated by other groups (23, 24). Besecke et al. (24) described a novel nonpeptide antagonist, A-198401, which binds to the human GnRH receptor with a 20- to 70-fold higher affinity than to the dog receptor. Furuya et al. (25) reported another class of nonpeptide GnRH antagonists that also had a higher affinity for the human receptor. Although these authors did not reveal the molecular basis for the species difference, it is possible that the same amino acid substitution (Phe313 to Leu313) we identified above is responsible for the decreased affinity for the dog receptor. If so, it suggests that Phe313 is a critical site for the binding of a variety of structural classes of nonpeptide ligands. Elucidation of the mechanism of nonpeptide ligand-receptor interaction will facilitate the rational design of improved GnRH antagonists.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Reagents
Dog pituitaries were obtained from Rockland Immunochemicals (Gilbertsville, PA). The TRIzol reagent and oligo(dT) cellulose were purchased from Promega Corp. (Madison, WI). SuperScript II reverse transcriptase, PCR kit, pBlueScript, Lipofectamine, regular tissue culture medium, inositol-free F12 media, FBS, and dialyzed FBS were purchased from Life Technologies, Inc. (Gaithersburg, MD). Site-directed mutagenesis kit and AG1-X18 columns were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA). Cetrorelix and the radio-labeled peptide ligand 5-(125Iodo-Tyr)-buserelin were obtained from Woods Assay (Portland, OR). 3H-myo-inositol was obtained from NEN Life Science Products (Boston, MA). GnRH and its synthetic analogs were obtained from Bachem (Torrance, CA). All primers used in this study were from Life Technologies, Inc. The restriction enzymes and T4 DNA ligase were purchased from Roche Molecular Biochemicals (Indianapolis, IN). All sequencing services were performed by ACGT, Inc. (Northbrook, IL). pCR2.1 and pcDNA3.1 plasmids were obtained from Invitrogen (San Diego, CA). [32P]-dCTP and random priming kits were from Amersham Pharmacia Biotech (Arlington Heights, IL). The Lambda DASH dog genomic library was purchased from Stratagene (La Jolla, CA). The nonpeptide GnRH antagonists used in this study were synthesized at Merck & Co., Inc. (Rahway, NJ) (Refs. 14, 15, 16 and R. J. Devita, M. Parikh, J. Jiang, M. T. Goulet, M. J. Wyvratt, J-L. Lo, Y. T. Yang, J. Cui, N. Ren, K. Cheng, and R. G. Smith, manuscript in preparation).

mRNA Isolation, cDNA Synthesis, and PCR
Total RNA from dog pituitaries (snap frozen in liquid nitrogen within 1–2 min of animals’ death) was prepared using the TRIzol reagents following the manufacturer’s instructions. Poly (A) RNA was isolated from total RNA by column chromatography on oligo (dT) cellulose. The yield of poly (A) mRNA from total RNA was approximately 0.5%.

First-strand cDNA was synthesized from poly (A)+ mRNA using SuperScript II reverse transcriptase as per the manufacturer’s instruction. One-tenth of the volume was used for each RT-PCR reaction.

Cloning and Sequencing of the Dog GnRH Receptor
The dog GnRH receptor was cloned by the following steps: 1) isolation of the genomic dog GnRH receptor; 2) cloning of partial cDNAs from the genomic clones by PCR using the degenerate primers; 3) cloning of the cDNA encoding the entire open reading frame (ORF) from the dog pituitary mRNA by RT-PCR using the specific primers.

Isolation of the Genomic Dog GnRH Receptor
The Lambda DASH dog genomic library was screened by a probe of approximately 1 kb cDNA encoding the ORF of the human GnRH receptor under low-stringency hybridization conditions. These include using 40% formamide in prehybridization and hybridization solutions and washing the blot at 50 C in 0.5x SSC and 0.1% SDS. A single positive clone was isolated after three rounds of plaque purification. Phage DNA was prepared, and the insert was subcloned into pBlueScript at SalI.

Cloning of Partial cDNAs from the Genomic Clones
Degenerate primers JC1 (in TM 1, 5'-ACTCGTCGACAAYCAYTGYAGYGCNATHAA-3') and JC3 (in TM 2, 5'-ACTCGAATTCTACCAYTGNACNGTDATRTTCCACATNCC-3') were used to clone the partial cDNA of exon 1 by PCR from the genomic clones. JC5 (in TM 6, 5'-ACTCGTCGACAARATGACNGTNGCNTTYGC-3') and JC8 (in TM 7, 5'-CTACAAAGAAAARTANCCRTADATNAGNGGRTC-3') were used to clone the partial cDNA of exon 3 from the genomic clones. The PCR products were individually subcloned into the plasmid vector pCR2.1, and the sequences of the cDNA insert were determined at both strands. Based on these results, specific primers were designed and used to sequentially determine the coding sequences of the entire ORF from the genomic clones. During this work, some DNA sequences immediately upstream of translation initiator ATG and downstream of stop codon TAA were also determined.2 The abbreviations used are: Y = C/T; n = A/C/T/G; H = A/T/C; D = A/T/G; r = A/G.

Cloning of the cDNA Encoding the Entire ORF
The full-length coding cDNA of the dog GnRH receptor was cloned from the pituitary mRNA by RT-PCR using primers Dog 6 (5'-ACTCGAATTCGCCACCATGGCAAGCGCCTCTCC-3') and Dog 7 (5'-ACTCTCTAGATTACAGAGAGAAATATCC-3'). The PCR product was subcloned into the expression vector pcDNA3.1. Four clones derived from independent PCRs had identical sequences.

Other GnRH Receptors
The cDNA encoding the human receptor was cloned from the human brain cDNA library by PCR using the primers Human 1 (5'-ATGCGAATTCGCCACCATGGCAAACAGTGCCTCTC-C-3') and Human 2 (5'-ATGCTCTAGATCACAGAGAAAAA-TATCCATAG-3'). The PCR product was subcloned into pcDNA3.1, and the integrity of the sequence was confirmed by DNA sequencing.

The rat GnRH receptor cDNA was kindly provided by Dr. Paul Liberator (Merck Research Laboratories, Rahway, NJ), and it was also subcloned into pcDNA3.1.

The GnRH receptors of human, monkey, dog, and rat were inserted at EcoRI and XbaI sites of pcDNA3.1. A Kozak sequence (GCCACCATGG) was added to each cDNA to optimize the protein expression.

Construction of the Chimeric Receptors
The chimera chi-1 was constructed by ligating the EcoRI–HindIII fragment of the dog GnRH receptor and the HindIII–XbaI fragment of the human receptor with pcDNA3.1 that was digested with EcoR and XbaI. The chimera chi-2 was constructed by ligating the EcoRI–PstI fragment of the dog receptor and the PstI–XbaI fragment of the human receptor with pcDNA3.1 that was digested with EcoRI and XbaI.

Site-Directed Mutagenesis
All mutants were made on pcDNA3.1containing the GnRH receptors of human or dog using the Bio-Rad Laboratories, Inc. Muta-Gene M13 in vitro mutagenesis kit, according to the manufacturer’s instructions. Altered sequences of the mutants with the point mutation were verified by DNA sequencing.

Transient Transfection of CHO-K1 Cells
CHO-K1 cells were seeded in 24-well plates at a density of 150,000 cells per well in {alpha}-MEM medium containing 10% FBS, 1% Pen/Strep, and 10 mM HEPES. Twenty-four hours after seeding, cells were transfected with 3 µg plasmid DNA by Lipofectamine in serum-free Optimem I medium for 5 h according to the manufacturer’s instruction. Transfected cells were cultured for an additional 24–40 h at 37 C before assays were performed. pcDNA3.1 carrying the wild-type or mutated GnRH receptor was used for transient transfection in whole-cell binding and phosphoinositide (PI) turnover assays.

Whole-Cell Binding Assay
Transfected cells were washed twice with a modified Medium 199 containing 0.1% fat-free BSA and 10 mM HEPES (pH 7.4). The radiolabeled peptide ligand 5-(125Iodo-Tyr)-buserelin at a final concentration of 0.1 nM (specific activity at 1000 Ci/mmol) was incubated with cells at 22 C for 1 h in the presence of various concentrations of a test compound. After the incubation, the cells were washed four times with 750 µl of cold PBS (pH 7.5), and dissolved in a buffer containing 0.2 N NaOH and 1% Triton X-100. The cell suspension was transferred into a tube and counted in a {gamma}- scintillation distometer.

The maximal binding (Bmax) was determined by Prism (GraphPad Software, Inc.) according to the equation Total binding = (Bmax[Hot]/[Hot] + [Cold] + Kd) + NS.

PI Turnover Assay
Transfected cells were washed twice with 0.5 ml of inositol-free F12 medium containing 10% dialyzed FBS, 1% Pen/Strep, and 2 mM glutamine, and incubated in 1 ml of F12 medium containing 3H-inositol (2 µCi) for 24 h at 37 C. After the incubation, cells were washed three times with 1 ml of PBS containing 10 mM LiCl and treated with various concentrations of an antagonist for 2 h before the addition of 1 nM GnRH, which is the approximate EC50 value of the wild-type human and dog GnRH receptors in response to GnRH stimulation. After incubation at 37 C for 1 h, the medium was removed, and the cells were lysed with 1 ml of 0.1 M formic acid. The plates were freeze-thawed once at -80 C, and the cell extract was applied onto a Dowex AG1-X8 column. The column was washed twice with 1 ml of water to remove the free 3H-inositol, and 3H-inositol phosphates were eluted three times with 1 ml of 2 M ammonium formate in 1 M formic acid. The eluate was then counted in a scintillation counter.


    ACKNOWLEDGMENTS
 
We thank Drs. John Kozarich and Matthew J. Wyvratt for helpful discussions and for critically reading the manuscript, and Dr. Paul Liberator for kindly providing the rat GnRH receptor clone. We gratefully acknowledge the assistance of Dr. Edward Hayes and Mr. Mike Dashkevicz for cell line maintenance.


    FOOTNOTES
 
Address requests for reprints to: Jisong Cui, Department of Endocrinology and Chemical Biology, Merck Research Laboratories, 126 East Lincoln Avenue, P.O. Box 2000, RY80T-126, Rahway, New Jersey 07065.

1 These sequence data have been submitted to the GenBank database under accession number AF206513. Back

2 These sequence data have been submitted to the GenBank database (nos. AF224076 and AF 223891). Back

Received for publication December 13, 1999. Revision received February 15, 2000. Accepted for publication February 17, 2000.


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