From the MRC/UCT Research Unit for Molecular
Reproductive Endocrinology and the ¶ Department of Medicine,
University of Cape Town, Observatory 7925, South Africa, ** MRC Human
Reproductive Sciences Unit, 37 Chalmers Street,
Edinburgh EH3 9ET, Scotland, United Kingdom,
§§ Division of Integrative Biology, Roslin
Institute (Edinburgh), Roslin, Midlothian EH25 9PS, United Kingdom,
and the ¶¶ Fishberg Research Center in Neurobiology, the
Mount Sinai Medical Center, New York, New York 10029
Received for publication, October 3, 2000, and in revised form, November 30, 2000
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ABSTRACT |
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Mammalian receptors for gonadotropin-releasing
hormone (GnRH) have over 85% sequence homology and similar ligand
selectivity. Biological studies indicated that the chicken GnRH
receptor has a distinct pharmacology, and certain antagonists of
mammalian GnRH receptors function as agonists. To explore the
structural determinants of this, we have cloned a chicken pituitary
GnRH receptor and demonstrated that it has marked differences in
primary amino acid sequence (59% homology) and in its interactions
with GnRH analogs. The chicken GnRH receptor had high affinity for mammalian GnRH (Ki 4.1 ± 1.2 nM)
, similar to the human receptor (Ki 4.8 ± 1.2 nM). But, in contrast to the human receptor, it also had
high affinity for chicken GnRH ([Gln8]GnRH) and GnRH II
([His5,Trp7,Tyr8]GnRH)
(Ki 5.3 ± 0.5 and 0.6 ± 0.01 nM). Three mammalian receptor antagonists were also pure
antagonists in the chicken GnRH receptor. Another three, characterized
by D-Lys6 or
D-isopropyl-Lys6 moieties, functioned as pure
antagonists in the human receptor but were full or partial agonists in
the chicken receptor. This suggests that the Lys side chain interacts
with functional groups of the chicken GnRH receptor to stabilize it in
the active conformation and that these groups are not available in the
activated human GnRH receptor. Substitution of the human receptor
extracellular loop two with the chicken extracellular loop two
identified this domain as capable of conferring agonist activity to
mammalian antagonists. Although functioning of antagonists as agonists
has been shown to be species-dependent for several GPCRs, the
dependence of this on an extracellular domain has not been described.
Gonadotropin-releasing hormone
(GnRH)1
(Glu(P)-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly10NH2)
is synthesized in hypothalamic neurons and released into the
hypothalamic-hypophysial portal system to regulate the synthesis and
release of pituitary gonadotropins that in turn regulate the function
of the testes and ovaries. Consequently, GnRH analogs are extensively
employed as therapeutic agents in sex hormone-dependent diseases (1-3). The GnRH receptor is a rhodopsin-like G
protein-coupled receptor (GPCR) (4). Progress has been made in defining
the structure of mammalian GnRH receptors (5-8) and in identifying amino acid residues that are important for ligand binding (9-12) and
coupling to G proteins (13, 14). Some insight has also been obtained
into the molecular entities involved in receptor activation. The
interaction of Asn2.50(87), in transmembrane domain 2 (TM2), with Asp7.49(318) in TM7 of the mouse GnRH receptor
appears to have a role in stabilizing the activated receptor
conformation (7, 8). In common with other GPCRs, the highly conserved
Asp-Arg (DR) sequence at the intracellular boundary of TM3 has an
integral role in activation of the GnRH receptor (15). Nevertheless,
the structural features that determine whether a ligand will interact
with the GnRH receptor as an agonist or antagonist, stabilizing the
active or inactive conformation of the receptor, are poorly understood.
The high amino acid sequence homology of the mammalian GnRH receptors
(over 85%) is paralleled by a similarity in pharmacological properties
(4), which has made comparative sequence analysis of conserved and
altered amino acids uninformative in identifying functionally important
residues. In contrast, nonmammalian GnRH receptors have distinctly
different pharmacology, both in ligand selectivity and G protein
coupling (4, 16-19). Biological assays indicate that the chicken GnRH
receptor exhibits well defined differences in ligand selectivity
compared with mammalian GnRH receptors (16). In particular, some
antagonists of mammalian GnRH receptors act as agonists of the chicken
GnRH receptor, stimulating luteinizing hormone (LH) release from
chicken pituitary cells (20). Cloning and characterization of the
chicken gonadotrope GnRH receptor would, therefore, potentially provide
the means for identifying domains and residues involved in ligand
selectivity and underlying receptor activation.
We report the cloning of a novel GnRH receptor from chicken pituitary
that differs from the mammalian GnRH receptor in its primary structure,
ligand selectivity, and in the agonistic behavior of certain mammalian
GnRH receptor antagonists. Analysis of the functional properties of a
range of antagonists of the mammalian GnRH receptors in the chicken
GnRH receptor shows that agonism in the chicken receptor is conferred
by a basic D-Lys or D-Ipr-Lys in position 6. Chicken-human chimeric receptors identified the receptor determinant of
the agonist activity of the "antagonist" peptides as extracellular
loop 2 (EC2) of the chicken GnRH receptor.
Reagents and Peptides--
The sequences of GnRH analogs used in
the study are shown in Table I. Agonists were synthesized by
conventional solid-phase methodology and purified to more than 95%
homogeneity by preparative C-18 reversed-phase chromatography (16).
Antagonist 26 was a gift from Dr. D. H. Coy (Tulane University
Medical Center, New Orleans, LA) and antagonists 27, 135-18, 135-25, 134-53, and 134-46 were from Dr. R. W. Roeske (Indiana University,
Indianapolis, IN). Other chemicals were obtained from Merck or Sigma.
Receptor Amino Acid Residue Numbering--
A numbering scheme,
in which amino acids of GnRH receptors are numbered relative to the
most conserved residues in the TM segments of the rhodopsin-like GPCRs,
is used to facilitate comparison among different receptors (21). The
amino acid identifier, which follows the name of the amino acid,
consists of the TM number followed by the position of the amino acid
relative to the most conserved residue in that TM, which is assigned
the number 50, and the sequence number of the amino acid in its
receptor, in parentheses. For example, the Asp residue that is located
immediately amino-terminal to the most conserved residue,
Pro7.50, in TM7 is designated Asp7.49(319) in
the human GnRH receptor and Asp7.49(310) in the chicken
GnRH receptor (see Fig. 2 for alignment of these receptor sequences).
Cloning of the Chicken GnRH Receptor Gene--
A 120-base pair
product encoding EC3 was amplified from 1 µg of chicken genomic DNA
using the degenerate primer pairs (JH5S/JH6A) to conserved regions in
TM6 and TM7 of GnRH receptors (22) (Fig. 1). The 120-base pair product
was cloned into the pMOS-blue vector (Amersham Pharmacia Biotech),
labeled with [
Cloning of Chicken GnRH Receptor cDNA--
Four antisense
primers were designed from the pCH1 nucleotide sequence (Fig. 1). Two
of these primers YS6, 5'-TCTAGTCTCCTTTTGGGTACATCTCTTC-3', and YS5,
5'-TGGGTACATCTCTTCAGCACACCGT-3', were designed to hybridize to
sequences of the 3'-untranslated region. Primers YS4,
5'-GGTGCATGTGTGCAGCAAACC-3', and YS3, 5'-GGGCATCCTCTGGATCATGGC-3', were
designed on the basis of the EC3 sequences of the receptor.
cDNA was synthesized from 2 µg of total RNA isolated from the
pituitaries of castrated chickens (Marathon cDNA synthesis kit, CLONTECH, Palo Alto, CA). The YS6 primer was used
to initiate first strand synthesis. Marathon cDNA adapters, which
contain the AP1 and AP2 primers, were ligated to the ends of the
cDNA. The 5'-end of the chicken GnRH receptor cDNA was
amplified by three rounds of nested PCR with KlenTaq polymerase
(CLONTECH), using the following combination of
primers: round 1 (AP1/YS5), round 2 (AP2/YS4), and round 3 (AP2/YS3).
Southern blot analysis of the PCR products identified three bands of
0.8, 0.9, and 1.2 kb that hybridized to
Two sense primers were designed to the region 5' to the start codon of
the chicken receptor as follows: YS1, 5'-GCTGAGCACTTGTGCTGCCT-3', and
YS2, 5'-CACTTGTGCTGCCTGACTTGCTG-3' (Fig. 1). Two rounds of nested PCR
with primer combinations (YS1/YS6 and YS2/YS5) yielded a ~1.2-kb band
from the castrated chicken pituitary DNA. This band was subcloned into
the pMOS vector. Two clones, pCH4 and pCH5, were isolated, and
nucleotide sequencing confirmed that they contained the entire open
reading frame of the chicken GnRH receptor. Comparison of the
nucleotide sequences of these clones with those of the genomic clones
identified a number of differences that might have arisen during the
PCRs or represented polymorphisms. The pCH4 clone showed nucleotide
substitutions resulting in amino acid substitutions of Gln for
Lys1.30(40) and Tyr for His1.65(75) compared
with the genomic sequences. The pCH5 clone contained Arg in place of
Cys5.23(189) of the genomic clone. Both clones showed a
substitution of Arg for Gln7.77(338). As it is uncertain
whether these differences represent polymorphism or PCR errors, three
different DNA fragments from the pCH4, pCH5, and pCH1 were ligated to
reconstruct a chicken GnRH receptor (cGnRH-R) encoding amino acid
sequences identical to those of the genomic clone. The
XbaI/SphI fragment from the pCH5 was ligated to
the SphI/EcoRI fragment of the pCH4 and subcloned
into the pSK+ vector, eliminating the amino acid substitutions at
positions 40, 75, and 189 (the XbaI and EcoRI
sites were in the pMOS vector). To eliminate the substitution at
position 338, the NotI/AvaI fragment from this
chimeric clone was ligated to the AvaI/PstI
fragment from pCH1 (NotI and PstI sites were in
the pSK+ vector) and subcloned into the pSK vector to yield the
full-length cGnRH-R. The NotI/XhoI fragment of
the cGnRH-R was subcloned into the mammalian expression vector
pcDNA I/AMP (Invitrogen, Carlsbad, CA). Sequencing of the resulting
chicken GnRH receptor clone verified that the encoded amino acid
sequence was identical to that of the genomic clone.
Construction of Chimeric Chicken-Human GnRH Receptor--
Silent
restriction endonuclease sites for BsrGI, StuI,
HpaI, and SnaBI were introduced at the
extracellular ends of TM4 to TM7 of the human GnRH receptor by
site-directed mutagenesis (24). The resulting receptor exhibited ligand
binding and IP accumulation that were indistinguishable from those of
the wild type human GnRH receptor. Chimeric GnRH receptors were
constructed by excising the extracellular loops of the human receptor
and replacing them with equivalent loops of the chicken GnRH receptor
(Fig. 2), using appropriate restriction sites. EC2 and EC3 of the
chicken GnRH receptor were generated by PCR amplification of the
chicken GnRH receptor using Deep Vent DNA polymerase (New England
Biolabs, Beverly, MA) and primers flanked by the appropriate
restriction endonuclease recognition sequences. Mutations were
confirmed by DNA sequencing.
Cell Culture and Receptor Transfection--
COS-1 cells, grown
in Dulbecco's modified Eagle's medium with 10% fetal bovine serum,
were seeded into 12-well plates (Corning Glass) (0.6-1 × 105 cells/25-mm well), pre-coated with
poly-D-lysine (10 µg/ml). Cells were transfected for
3.5 h with plasmid DNA (0.5 to 1 µg/well), using a modified
DEAE-dextran method (25).
Inositol Phosphate (IP) Assay--
IP production was measured as
described previously (26). Briefly, cells were labeled with
myo-[3H]inositol (1 µCi/ml, Amersham
Pharmacia Biotech) in Medium 199, supplemented with 2% fetal bovine
serum, penicillin, and streptomycin for 16-22 h. Cells were washed
twice with buffer A (140 mM NaCl, 4 mM KCl, 1 mM CaCl2, 1 mM MgCl2,
20 mM HEPES, 0.1% bovine serum albumin, and 8 mM D-glucose, pH 7.4) and incubated for 1 h at 37 °C with 10 mM LiCl and appropriate
concentrations of GnRH agonist or with antagonist in the presence and
absence of 1 or 10 nM [Gln8]GnRH (chicken
GnRH receptor) or 1 nM mammalian GnRH (human GnRH receptor). Experiments were performed in duplicate and repeated at
least three times.
Radioligand Binding Assay--
Radioligand binding assays were
performed on intact cells 48 h after transfection. Transfected
cells in 12-well culture plates were washed and incubated for 3 h
at 4 °C with 125I-GnRH-A (100,000 cpm) and various
concentrations of unlabeled GnRH agonists or antagonists in buffer A as
previously described (14, 27). Nonspecific binding was determined in
the presence of 1 µM unlabeled antagonist 27. After
incubation, the cells were washed three times and solubilized in 0.5 ml
of 0.1 M NaOH, and the radioactivity was counted. All
experiments were performed in triplicate and repeated at least twice.
Data Analysis--
Ki, ED50
(peptide concentration required for half-maximal IP formation), and
IC50 (antagonist concentration required to half-maximally
inhibit IP formation) values were calculated by nonlinear regression
analysis using the PRISM program (GraphPad Inc., San Diego, CA).
Cloning of Chicken GnRH Receptor cDNA--
Screening of
chicken pituitary cDNA libraries failed to identify any GnRH
receptor clones. As the chicken thyrotropin-releasing hormone receptor
was cloned from the same libraries (27), it appears that GnRH receptor
mRNA is expressed at low levels in chicken pituitaries, as in some
mammalian species (28, 29). The full-length chicken GnRH receptor
cDNA was therefore isolated by the combined strategies of genomic
library screening, 5'-rapid amplification of cDNA ends, and PCR of
total RNA prepared from pituitaries of castrated chickens (see under
"Experimental Procedures"). Sequencing of genomic clones revealed
that the chicken GnRH receptor has introns located in TM4 and IC3 (Fig.
1) as in mammalian GnRH receptors (30,
31). But, unlike the mammalian receptors, an additional intron is
present in the amino-terminal domain (Fig. 1). Full-length chicken GnRH
receptor cDNA clones obtained by PCR varied in the coding of the
amino acids Lys1.30(40), His1.65(75),
Cys5.23(189), and Gln7.77(338) (see
"Experimental Procedures"). Since it was uncertain whether these
were errors incorporated by PCR or polymorphisms, a "wild type"
chicken GnRH receptor cDNA, which corresponded to the genomic sequence, was constructed (Fig. 1) and used for all of the experiments described.
The cloned chicken GnRH receptor cDNA encodes a 375-amino acid
polypeptide with the seven hydrophobic putative TM domains, connected
by three cytosolic and three extracellular loops, extracellular amino-terminal and cytosolic carboxyl-terminal domains that are characteristic of GPCRs (Fig. 2). The
chicken GnRH receptor has low amino acid sequence identity (41%) and
homology (59%) with the human and other mammalian GnRH receptors,
excluding the highly variable amino-terminal domain and the
carboxyl-terminal tail that is absent from mammalian GnRH receptors (4)
but present in other nonmammalian GnRH receptors (17-19). The
carboxyl-terminal domain has been shown to regulate GnRH receptor
desensitization and internalization (32, 33). Also similar to other
nonmammalian GnRH receptors, the chicken GnRH receptor has Asp in both
loci of the conserved helix 2/helix 7 functional microdomain. This microdomain consists of
Asn2.50(87)/Asp7.49(319) in the human GnRH
receptor and Asp2.50/Asn7.49 in most other
GPCRs, and it regulates GPCR coupling and expression (7, 8). The
presence of two Asp residues in the chicken GnRH receptor suggests that
the nonmammalian GnRH receptors may represent an evolutionary
intermediate between the Asp2.50/Asn7.49
arrangement of the microdomain found in most GPCRs and the
Asn2.50/Asp7.49 of the mammalian GnRH
receptors. The presence of Asp2.50/Asn7.49 in
the Drosophila melanogaster homolog of the GnRH receptor
(34) supports this conclusion. Other residues that are important for coupling of mammalian GnRH receptors to cytosolic signal transduction, the Arg cage motif (DRXXX(I/V)) at the cytosolic end of TM3
(15) and Ala5.29(261) in IC3 (14) are conserved in the
chicken GnRH receptor. All of the residues previously shown to have a
role in ligand binding of mammalian GnRH receptors,
Asp2.61(98) (12), Asn2.65(102) (9),
Lys3.32(121) (11), and Glu7.32(301) (10), are
conserved in the chicken receptor (Fig. 2).
GnRH Agonist Interactions--
The cloned chicken GnRH receptor
exhibited high affinity binding to a series of GnRH agonists (Table
II). It had high affinity for both mammalian GnRH and the native
chicken ligand [Gln8]GnRH (Ki 4.1 ± 1.2 and 5.3 ± 0.5 nM, respectively), in contrast
to the human GnRH receptor, which had similar high affinity for
mammalian GnRH (Ki 4.8 ± 1.2 nM)
but low affinity for [Gln8]GnRH (Ki
174 ± 69 nM) (Table II). The chicken GnRH receptor had much higher affinity for GnRH II (0.60 ± 0.01 nM), also contrasting with low affinity in the human GnRH
receptor (Ki 39 ± 8.5 nM). The
substitution of a D-amino acid for Gly6 in GnRH
is thought to constrain the peptide in the biologically active
The cloned chicken GnRH receptor stimulated IP accumulation in response
to GnRH agonists (Table II). The rank order of agonist ED50
values was the same as the rank order of Ki values determined in ligand binding assays (Table II). The
Ki values for binding of these agonists to the
cloned chicken GnRH receptor and the ED50 values for
agonist-stimulated IP accumulation are consistent with the
ED50 values previously reported for stimulation of LH
release from cultured chicken pituitary cells (16, 35). Ligand-independent IP accumulation was not detected in cells
transfected with the chicken GnRH receptor.
Agonism of Mammalian Receptor Antagonists--
We have previously
reported that some antagonists of mammalian GnRH receptors behave as
agonists, stimulating LH release from cultured chicken pituitary cells
(20). A series of mammalian receptor antagonists was used to define the
structural basis of agonism in the cloned chicken pituitary. All of the
analogs functioned as full antagonists with high binding affinity in
the human GnRH receptor (Table II and Fig.
3). The analogs had much lower binding affinity for the cloned chicken GnRH receptor (Table II). Furthermore, three analogs exhibited distinct agonist activity, stimulating IP
accumulation in cells expressing the cloned chicken GnRH receptor (Table II and Fig. 3), whereas the other three analogs functioned as
pure antagonists (Table II). Antagonist 135-18 was a full agonist, and
antagonists 135-25 and 26 were partial agonists (Fig. 3). All of the
analogs that showed agonist activity contained a basic D-amino acid (D-Lys or D-Ipr-Lys)
in position 6, and all peptides that were antagonists in the chicken
GnRH receptor had uncharged side chains in this position (Table
I).
The agonist behavior of three antagonists in the chicken receptor may
arise from interactions of these peptides with amino acid residues that
are unique to the chicken receptor. We attempted to identify the
domains of the chicken receptor that are involved using chimeric
receptors in which EC domains of the human receptor were substituted
with EC domains of the chicken receptor. Since EC1 is highly conserved
(Fig. 2), exchanges were confined to EC2 and EC3. The chimeric receptor
containing EC3 of the chicken GnRH receptor did not bind GnRH or
stimulate IP accumulation in response to GnRH, suggesting that the EC3
chimera was poorly expressed or uncoupled from activation of
phospholipase C (data not shown). However, the chimera containing the
chicken receptor EC2 substituted in the human receptor exhibited high
affinity binding and IP accumulation. The EC2-containing chimera bound
antagonists 26, 135-25, and 135-18 with high affinities
(Ki 7.8 ± 2.9; 12 ± 4.7; and 18 ± 5.3 nM, respectively), which were similar to their
affinities for the wild type human receptor and higher than those for
the chicken receptor (Table II),
indicating that the NH2-terminal domain, EC1, EC3, and
superficial regions of the TM domains of the human receptor are major
contributors to high affinity building of the antagonists (Tables II).
Antagonists 26 and 132-25, which were partial agonists in the chicken
GnRH receptor, behaved as antagonists in the chimera (Fig.
4), similar to the wild type human
receptor. Antagonist 135-18, which was a full antagonist in the human
GnRH receptor and a full agonist in the chicken GnRH receptor,
exhibited partial agonist behavior in the chimera (Fig. 4). As was
found for the chicken GnRH receptor, no constitutive activity was
detectable in the chimeric receptor.
The definitive molecular delineation of ligand binding, signal
propagation, and G protein coupling of the human GnRH receptor and the
development of GnRH analogs is a major goal in reproductive medicine.
Progress in this regard has been made through the cloning of GnRH
receptors and a combination of molecular modeling and mutagenesis
studies (4). Since the various mammalian receptors have close sequence
homology and similar ligand selectivity (4), information on the primary
sequence of a related, but pharmacologically distinct, nonmammalian
receptor would potentially contribute in these endeavors. The chicken
GnRH receptor that we have cloned exhibits marked pharmacological
differences in its interaction with GnRH agonist and antagonist analogs
and has sequence differences from the mammalian receptors that may be
used to identify functional residues.
Agonist-binding Site Differs in Chicken and Mammalian GnRH
Receptors--
The conservation of the Asp2.61(98),
Asn2.65(102), and Lys3.32(121)
residues in the chicken GnRH receptor is expected, as these residues
are believed to interact with the amino-
(Glu(P)1-His2) and carboxyl-terminal
(Gly10-NH2) residues of the GnRH ligands,
which are conserved in the native mammalian and chicken forms of GnRH
(9, 11, 12). As expected from biological assays, the chicken GnRH
receptor does not distinguish between its cognate native ligand,
[Gln8]GnRH (Ki 5.3 ± 0.5 nM), and mammalian GnRH (Ki 4.1 ± 1.2 nM). Surprisingly, the affinity of the chicken receptor for both ligands was as high as the affinity of the human GnRH receptor
which is selective for mammalian GnRH (Ki 4.8 ± 1.2 nM) and binds [Gln8]GnRH with low
affinity (Ki 174 ± 69 nM). The
high affinity of the chicken receptor is unexpected because high
affinity binding of mammalian GnRH depends on an interaction of
Arg8 with an acidic residue in the EC3 domain
(Glu3.32(301) in the mouse and Asp3.32(302) in
the human) (10). The equally high affinity of mammalian GnRH and
[Gln8]GnRH binding to the chicken receptor suggests,
therefore, that the
Arg8-Glu3.32(301)/Asp3.32(302)
interaction does not occur but is compensated by alternative interaction(s) in the chicken receptor. Thus, it appears that, although
there are interactions that are common to the mammalian and chicken
receptors, the binding sites differ in unique interactions.
This suggestion that [Gln8]GnRH utilizes a different
binding site in the chicken GnRH receptor is not unexpected.
[Gln8]GnRH is not configured in the folded Mammalian Antagonists Function as Agonists in the Chicken GnRH
Receptor--
The six mammalian GnRH receptor antagonists studied had
decreased affinities for the chicken GnRH receptor compared with the human receptor, emphasizing the differences in the ligand-binding site.
Three of the selected mammalian GnRH receptor antagonists were also
pure antagonists in their interaction with the chicken GnRH receptor.
Another three pure mammalian antagonists, 26, 135-25, and 135-18, behaved as partial or full agonists in their interaction with the
chicken GnRH receptor. There was no correlation of agonist/antagonist behavior and binding affinities at the chicken GnRH receptor (Table II).
Analysis of the sequences of the antagonist analogs revealed that a
single feature is unique to those with agonistic activity at the
chicken receptor. All three analogs with agonist activity have a basic
D-amino acid substitution (D-Lys or
D-Ipr-Lys) for Gly6 (Table I). Antagonists
135-18 and 135-25, which are full and partial agonists, respectively,
differ only in the presence of Ile and 1-MePal in position 5. This
suggests that the larger aromatic side chain (1-MePal) prevents 135-25 from acting as a full agonist in the chicken receptor. This conclusion
is supported by the partial agonism of antagonist 26 that also has a
large aromatic side chain (Tyr) in position 5. It appears that the
large aromatic side chain changes the orientation of the adjacent
D-Lys moiety such that its ability to interact with a
cognate receptor amino acid is impaired, thus decreasing agonist activity.
EC2 of Chicken GnRH Receptor Is a Determinant of Agonist Behavior
of Antagonists--
Insertion of the chicken GnRH receptor EC2 domain
into the human receptor conferred partial agonism to antagonist 135-18, which was a full agonist in the chicken receptor. Antagonists 135-25 and 26, which were partial agonists in the chicken receptor, had no
agonistic activity in the chimera. Thus, although the chicken EC2
domain is a determinant of agonist activity of antagonist 135-18, it is
insufficient to confer the same degree of agonist activity as is found
in the complete chicken receptor. This suggests that a combination of
appropriate domains is required or that the molecular dynamics of the
transition between active and inactive states differ in the human and
chicken receptors such that the interaction of antagonist 135-18 with
the EC2 chicken-human chimera cannot adequately stabilize the active conformation.
Proposed Mechanism of Agonist Activity of
Antagonists--
Contemporary thinking proposes that agonist analogs
bind and stabilize the receptor in the active conformation, antagonists bind both the active and inactive states of the receptor, and inverse
agonists bind and stabilize the inactive form (38). According to this
hypothesis, antagonist analogs, in binding both inactive and active
conformations of the receptor, would not disturb the equilibrium
between both forms; agonists would drive the equilibrium in favor of
the active conformation, and inverse agonists would drive the
equilibrium in favor of the inactive conformation. The agonist activity
of mammalian GnRH receptor antagonists at the chicken receptor is
therefore interpreted as resulting from a structural alteration that
confers a preference for binding to the active receptor conformation.
D-Lys6 or D-Ipr-Lys6 of
these antagonists appears to interact with a functional group, which is
available only in the chicken receptor in the active conformation.
These analogs can therefore stabilize the active conformation of the
chicken receptor but not the active form of the mammalian receptor.
Since a D-Lys or D-Ipr-Lys residue in position
6 of the mammalian GnRH receptor antagonists is the unique feature
associated with agonist activity, it presumably interacts with a
receptor amino acid side chain that is accessible in the active
conformation of the chicken receptor and is absent from, or
inaccessible in, the active conformation of the human GnRH receptor. A
candidate for a strong interaction with D-Lys6
would be an acidic residue in the receptor. Since substitution of the
chicken EC2 in the human receptor conferred partial agonism to
antagonist 135-18, the Glu5.34(200) and
Glu5.35(201) residues in the chicken EC2
(His5.34(207) and Gln5.35(208) in mammalian
receptors) are candidates. Glu5.35(201) is also present in
the Xenopus GnRH receptor in which antagonist 135-18 also
behaves as an agonist.2
An alternative proposal is that ligand binding induces the active state
of the receptor (39). In the
Mutations in a number of GPCRs have been described that confer agonism
to antagonists. Mutations in TM3 of the AT1 angiotensin receptor (39,
42), rhodopsin (43), and the
In conclusion, our findings here identified a single amino acid side
chain (D-Lys6 or
D-Ipr-Lys6) in mammalian GnRH receptor
antagonists that confers agonist activities to these antagonists when
interacting with the chicken GnRH receptor. This phenomenon is
conferred to the human receptor with incorporation of EC2 of the
chicken GnRH receptor. This suggests that an interaction of the
D-Lys6 or D-Ipr-Lys6
side chain with a residue in EC2 of the chicken GnRH receptor stabilizes the active conformation of the receptor. Identification of
other contact sites of this ligand will assist in delineating molecular
distances of TM domains of the GnRH receptor in the active and inactive
conformations and shed light on the molecular mechanism of
ligand-mediated receptor activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
32P]dCTP (Amersham Pharmacia Biotech)
by random priming using the Megaprimer labeling system (Amersham
Pharmacia Biotech), and used to screen a
Charon 4A chicken genomic
library (23). Three positive clones were isolated after tertiary screening.
DNA was purified from one of these clones (cGnRH-R.g-22). Southern
blot analysis of a PstI digest of cGnRH-R.g-22 identified a
2.7-kb fragment that hybridized with the 120-base pair probe used to
screen the library. The 2.7-kb fragment (pCH1) was cloned into pSK+
(Bluescript, Strategene, La Jolla, CA) and partially sequenced. The
pCH1 clone contained the entire 3'-coding region of the GnRH receptor
gene, including an intron within IC3 (Fig. 1). Partial nucleotide
sequence analysis of PstI fragments cloned into the pSK+
vector from the GnRH-R.g-22 identified a clone (pCH2) encoding the
region of the cGnRH-R from the 4th amino acid of the amino-terminal
region to the middle of TM4. The pCH2 construct was verified to contain
partial sequences of two introns at each end (Fig. 1).
32P-labeled
pCH2. These bands were subcloned into the pMOS-blue vector. One clone,
pCH3, was sequenced and shown to consist of the 5'-untranslated region
of chicken GnRH receptor cDNA extending to the YS3 primer (Fig.
1).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic diagram illustrating the cloning
and construction of the chicken GnRH receptor cDNA
(cGnRH-R). The designed primers and restriction
enzyme sites are indicated. Various amino acid differences from the
genomic clones are shown in the two full-length cGnRH-R cDNA
clones. ORF, open reading frame; The transmembrane domains
are indicated by hatching; *, stop codon; introns are shown
as lines.
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[in a new window]
Fig. 2.
Alignment of the deduced amino acid sequences
of the cloned chicken and human GnRH receptors. Amino acid numbers
are indicated for the chicken GnRH receptor above the sequence and at
the left side for both receptors. Sequences that are
conserved in both receptors are shaded. The putative TM segments, EC
and IC loops are assigned according to those predicted for the human
receptor (4) and TM segments are indicated by double bars
above the chicken GnRH receptor sequence; period, space.
Human GnRH receptor sequences that were substituted with chicken GnRH
receptor sequences in the chimeric receptors are indicated by
bars below the human receptor sequence.
-II-turn conformation and enhance binding affinity for mammalian
GnRH receptors (see Ref. 4). This is reflected in the 3-fold
enhancement of the affinities of [D-Arg6]GnRH
II and GnRH-A compared with unconstrained ligands in the human GnRH
receptor (Table II). In contrast, there was little or no enhancement in
binding affinity of the D-amino acid-containing ligands for
the chicken GnRH receptor (Table II). These direct receptor binding
studies confirm previous bioassay data and support the suggestion that
incorporation of D-amino acids in position 6 of GnRH does
not enhance binding affinity for the chicken GnRH receptor and that
GnRH binds the chicken GnRH receptor differently from the mammalian
receptor (16).
View larger version (28K):
[in a new window]
Fig. 3.
Agonist activity of mammalian GnRH receptor
antagonists in the chicken GnRH receptor. IP formation was
measured in COS-1 cells transfected with the chicken GnRH receptor
(left panel) or the human GnRH receptor (right
panel) in the presence of antagonists 135-18 (A),
135-25 (B), and 26 (C) with (open
symbols) or without (filled symbols)
[Gln8]GnRH (1 or 10 nM for antagonist 135-25, left panel) or mammalian GnRH (1 nM, right
panel). Data are mean ± S.E. of experiments performed two or
three times in duplicate. Ten nM [Gln8]GnRH
was used with antagonist 135-25 to demonstrate antagonist activity as
this antagonist had high intrinsic agonist activity, and its
antagonistic activity could only be demonstrated with this higher dose
of agonist.
Primary structures of GnRH agonists and antagonists
Receptor binding and peptide-regulated IP production of GnRH analogs in
COS-1 cells expressing chicken and human GnRH receptors
View larger version (23K):
[in a new window]
Fig. 4.
Ability of antagonists to inhibit GnRH
stimulation of IP production or stimulate IP in COS-7 cells transiently
transfected with the human GnRH receptor chimera containing EC2 of the
chicken receptor. IP formation was measured in COS-1 cells
transfected with the EC2 chicken-human chimeric GnRH receptor incubated
with antagonists 135-18 (A), 135-25 (B), and 26 (C) in the absence ( ) or presence (
) of mammalian GnRH
(10 nM). Data are the mean ± S.E. of experiments
performed in duplicate and repeated three or four times.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
II-turn
conformation characteristic of mammalian GnRH (36, 37), and
substitution of a D-amino acid for Gly6, which
enhances the folded conformation, does not enhance binding affinity for
the chicken receptor as it does in the human receptor (Table II). The
amino- and carboxyl-terminal residues, Glu(P)1,
His2, and Gly10NH2, of the ligand
and their cognate mammalian receptor binding residues,
Asp2.61(98), Asn2.65(102), and
Lys3.32(121), are all conserved in species of fish,
amphibians, birds, and mammals (4, 17-19). We therefore propose that
the interactions between these residues are also conserved. Since the
receptors have different requirements for ligand conformation, we
further propose that the spatial arrangement of the receptor-binding
sites differs between the chicken and mammalian receptors. This allows the accommodation of binding of the configured ligand in mammalian receptors and the nonconfigured ligands in the chicken GnRH receptor, through the same interactions. This proposal suggesting that the receptors are configured differently is supported by the presence of
two Asp residues (Asp2.50(87) and Asp7.49(310))
in the helix 2/helix 7 functional microdomain of the wild type chicken
GnRH receptor, an arrangement that is not tolerated in the mammalian
GnRH receptor (7, 8).
-adrenergic (40) and 5HT2A (41)
receptors, a different positioning of certain ligands alters their
ability to induce the active state of the receptor. It may be conceived
that the interaction of D-Lys6 or
D-Ipr-Lys6 with extracellular EC2 of the
chicken receptor changes positioning of these ligands to allow
interaction with receptor-activating sites.
-adrenergic receptor (40), in TM4 of
µ- and
-opioid receptors (44), in TM6 of the µ-opioid receptor
(45), in IC3 of the
2A-adrenergic receptor (46), and in
IC2 of the V2 vasopressin receptor (47) conferred agonistic activity to
antagonist ligands. The present study is the first description of a
mutation in an extracellular domain that confers agonism to an
antagonist. Receptor activation is believed to involve movement
(e.g. rotation) of the TM domains and that this is
propagated into structural changes in the connecting IC loop domains
(reviewed in Refs. 48 and 49). Our demonstration that mutation of EC2
allows the interaction with a single residue in an antagonist to confer
agonism indicates that a distinct relationship exists between EC and TM
domains and that ligand interaction with EC domains can stabilize the
receptor in the active conformation. Indirect evidence that molecular
interactions with EC domains can lead to receptor activation has been
obtained from antibody studies. Antibodies against EC2 of M1 and M2
muscarinic, AT1 angiotensin, and
1- and
2-adrenergic receptors caused receptor activation (50-53), pointing to a role for this domain in stabilizing receptor conformation.
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FOOTNOTES |
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* This work was supported by the Medical Research Council and National Research Foundation (South Africa), the University of Cape Town, the British Council, the Medical Research Council (UK), the Biotechnology and Biological Sciences Research Council (Competitive Strategic Grant), and National Institutes of Health Grant RO1.DK46943.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ304414.
§ Current address: Division of Genomics and Bioinformatics, Roslin Institute, Roslin, Midlothian EH23 9PS, UK.
Current address: Dept. of Biochemistry, University of Cape
Town, Rondebosch 7700, South Africa.
Current address: Dept. of Biochemistry, University of
Stellenbosch, Matieland 7602, South Africa.
To whom correspondence should be addressed: MRC Human
Reproductive Sciences Unit, 37 Chalmers Street, Edinburgh EH3 9ET,
Scotland, UK. Tel.: 131 229 2575; Fax: 131 228 5571.
Published, JBC Papers in Press, December 8, 2000, DOI 10.1074/jbc.M009020200
2 T. R. Ott, B. E. Troskie, R. W. Roeske, C. A. Flanagan, N. Illing, and R. P. Millar, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: GnRH, gonadotropin-releasing hormone; EC, extracellular loop; GPCR, G protein-coupled receptor; IC, intracellular loop; IP, inositol phosphates; Ipr, isopropyl; LH, luteinizing hormone; PCR, polymerase chain reaction; TM, transmembrane segment; kb, kilobase pair.
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