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
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ABSTRACT
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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
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INTRODUCTION
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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 12 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.
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RESULTS
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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. 1
). 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. 1
), 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 17 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.
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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 1
).
However, the IC50 values determined using the
cells expressing the dog receptor were approximately 50- to 300-fold
higher (Table 1
). Figure 2A
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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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. 2B
),
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 2
), indicating that the dog receptor does
contain the critical sites for binding these peptide ligands. Figure 3
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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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 ( ) 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.
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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. 4A
). 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. 4A
). 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. 4B
). 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. 4C
). 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.
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. 5
. 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.
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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. 6
, 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. 6
, 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. 6A
). 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 ( ), h-R227T ( ),
h-R313L ( ), or d-R313F ( ) 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.
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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. 6C
). 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. 6C
) 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. 6
). 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. 7
, 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 (
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. 1 ) 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.
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DISCUSSION
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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. 7
, 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
NH2 of
Lys121 (Fig. 8
).
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. 2B
).

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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. 7
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.
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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.
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MATERIALS AND METHODS
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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 12 min of animals death) was prepared using the TRIzol
reagents following the manufacturers 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
manufacturers 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
EcoRIHindIII fragment of the dog GnRH receptor
and the HindIIIXbaI fragment of the human
receptor with pcDNA3.1 that was digested with EcoR and
XbaI. The chimera chi-2 was constructed by ligating the
EcoRIPstI fragment of the dog receptor and the
PstIXbaI 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
manufacturers 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
-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 manufacturers instruction. Transfected cells were cultured for an
additional 2440 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
- 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. 
2 These sequence data have been submitted to the
GenBank database (nos. AF224076 and AF 223891). 
Received for publication December 13, 1999.
Revision received February 15, 2000.
Accepted for publication February 17, 2000.
 |
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