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Medical Research Council Research Unit for Molecular Reproductive Endocrinology (B.E.T.), Department of Medical Biochemistry, University of Cape Town, Observatory 7925; Medical Research Council Reproductive Biology Unit (R.P.M.), Center for Reproductive Biology, Edinburgh, United Kingdom EH3 9ET; Department of Biochemistry (J.P.H)., University of Stellenbosch, Matieland 7602; and Department of Biochemistry (N.I.), University of Cape Town, Rondebosch 7700, South Africa
Address all correspondence and requests for reprints to: Dr. B. E. Troskie, Medical Research Council Research Unit for Molecular Reproductive Endocrinology, Department of Medical Biochemistry, University of Cape Town, Observatory 7925, South Africa. E-mail: Brigitte{at}Curie.uct.ac.za
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The pituitary GnRH receptor plays an important role in the regulation of reproductive function in vertebrates by integrating signals from the hypothalamus, with the release of gonadotropins from the pituitary (16, 17). GnRH analogs are currently used for the treatment of a wide range of reproductive disorders. A detailed knowledge of GnRH receptor/ligand interaction and the mechanisms of receptor activation would be useful in the development of new therapeutic agents. The cloning and analysis of the highly conserved mammalian GnRH receptors has been important for the delineation of amino acid residues important in ligand/receptor interactions (18). These studies used a combination of receptor mutagenesis, molecular modeling, and intuitive insights based on receptor/ligand specificity of other G protein-coupled receptors (GPCRs) (18). As expected of GPCRs that bind to small peptide hormones (<40 amino acids) (19), several amino acid residues required for GnRH recognition and binding have been identified in the superficial transmembrane (TM) domains and the extracellular loops (18). An alternative approach to studying ligand-receptor interactions is the observation of coordinated structural and functional changes in evolutionarily distinct GnRH receptors that respond to the same endogenous GnRHs. Amino acid residues or motifs that are conserved in these receptors may be important for the overall conformation of the receptor, ligand binding, or G protein interactions. Residues that are not conserved may be unimportant or may contribute to unique receptor features, such as the difference between mammalian and nonmammalian GnRH receptor ligand specificities. An example of this is the requirement of the mammalian GnRH receptor for a positively charged arginine in position 8 of GnRH for high affinity binding (20). Nonmammalian GnRHs, which lack the charged residue in position 8, have been shown to be much less active in stimulating mammalian GnRH receptors (21). In contrast, the nonmammalian GnRH receptors that have been characterized to date have all been shown to be far less selective for mGnRH compared with other nonmammalian GnRHs (22, 23). However, unlike amphibians, none of these species has mGnRH as the natural form of GnRH regulating the pituitary-gonadal axis. We have, therefore, cloned and pharmacologically characterized the type I GnRH receptor cDNA (XLGnRH-R I) from Xenopus laevis to determine its ligand selectivity and its structural relationships to mammalian and nonmammalian GnRH receptors that also have mGnRH as their natural ligand in the pituitary.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Isolation of the amphibian GnRH receptor gene and cDNA
Genomic DNA was extracted from five livers. Two 120-bp products
corresponding to extracellular loop III were amplified from X.
laevis genomic DNA, using the degenerate PCR primers JH5s and JH6a
(Fig. 1
), which were designed to anneal
to conserved regions in the mammalian GnRH receptors (15, 23). One of
these had greater homology with the mammalian pituitary GnRH receptor,
and the other was a putative type II receptor (15). The putative type I
product was cloned into pCRII (Invitrogen, San Diego, CA;
pGXL1) and used as a probe to screen a X. laevis genomic DNA
library (Dr T. Sargent, NIH, Bethesda, MD). DNA from 12 positive
plaques was extracted and analyzed by restriction enzyme digestion
followed by Southern blotting. Plasmid pGXL3 (Fig. 1) was constructed
by subcloning the EcoRI restriction fragment containing the
region that hybridized to the EL3 probe into pBluescript SK(-)
(Stratagene, La Jolla, CA) and sequenced (T7 Sequenase
kit, version 2.0, Amersham Pharmacia Biotech, Arlington
Heights, IL). Nested gene-specific primers, XL2s
(5'-cagcctgagatgatcaacc-3'), XL3a (5'-gactgtggttaaggtactcagg-3'), and
XL4a (5'-ggtgtgcagcagaccaaagagg-3') were designed to anneal to the
sequence of pGXL1 (Fig. 1).
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-32P]dATP-labeled XL2s
primer. Positive products in the expected size range were subcloned
using the pMOS-blue cloning system (Amersham Pharmacia Biotech) and sequenced. The nucleotide sequences of plasmids
pCXL4 and pGXL3 were used to design primers to the 5'- and
3'-untranslated regions, respectively (XL1s,
5'-tgtgtcacagatatgagctac-3'; XL5a, 5'-tgctaggtcgatatgtagatc-3').
The full-length cDNA clone was amplified from pituitary cDNA and cloned
into pCDNA1/Amp (Invitrogen; pCXL5; Fig. 1
). Five
individual cDNA clones were sequenced in both directions. The structure
of the genomic clones were analyzed by PCR with gene-specific primers
designed to the cDNA sequence. PCR products were subcloned using the
pMOS-blue cloning system (Amersham Pharmacia Biotech) and
sequenced to determine the intron/exon boundaries.
GnRH receptor expression
RNA was extracted from several tissues, and first strand cDNA
was synthesized from 1 µg total RNA using Stratascript reverse
transcriptase (Stratagene), primed with an
oligo(deoxythymindine) cDNA synthesis primer for 10 min at room
temperature followed by 1 h at 42 C. The second strand cDNA was
synthesized using ribonuclease H, DNA polymerase I, and
T4 DNA ligase (all from Stratagene)
for 2.5 h at 16 C. The double stranded cDNA was precipitated with
2 M ammonium acetate and resuspended in 10 µl water. The
quality of the cDNA was assessed by running 2 µl on a 1.2% agarose
gel. cDNA was amplified by 35 cycles of PCR using primers to either the
X. laevis GnRH receptor sequence (XL1s and XL5a) or the
mouse ß-actin cDNA (sense primer, 5'-cattgttaccaactgggacgaca;
antisense primer, 5'-gctcggtcaggatcttcatgagg), which would amplify a
365-bp product from the cDNA. A negative control, where no cDNA was
added, and positive controls, where mouse ß-actin cDNA or pCXL5 cDNA
clones were used as template for the PCR, were also included. Products
were blotted onto nitrocellulose and probed with an appropriately
[-32P]dATP-labeled internally nested
primer.
GnRH receptor activity
DNA for transfection was prepared using the Wizard DNA
purification system (Promega Corp., Madison, WI). COS-1
cells were plated at an appropriate density (2 x
105 cells/well for inositol phosphate
assays and 4 x 105 cells/well for binding
assays) on poly-D-lysine-coated 12-well plates
(Corning, Inc., Corning, NY), 24 h before
transfection, in DMEM containing 10% FCS, penicillin (0.2 U/ml), and
streptomycin sulfate (100 mg/ml; Life Technologies, Inc.,
Gaithersburg, MD). DNA (2 µg) was transfected in serum-free DMEM for
4 h using an adapted diethylaminoethyl-dextran method (25), with a
50-min chloroquine (200 mM) treatment and a 2-min 10%
dimethylsulfoxide shock (26). Transfected cells were grown for 48
h before each experiment. Whole cell competitive binding assays were
performed as previously described (27). Peptides
([D-Ala6,N-Me-Leu7,Pro9-NHEt]GnRH,
antagonist 26,
[His5,D-Arg6,Trp7,Tyr8]GnRH,
and
[His5,D-Tyr6]GnRH)
were radioiodinated according to an adapted chloramine-T method (28, 29). Binding to untransfected cells was used to determine nonspecific
binding. Transfected COS-1 cells were labeled overnight in 0.5 ml
medium 199 (Life Technologies, Inc.) containing 2%
FCS and 2 mCi/ml myo-[2-3H]inositol
(Amersham Pharmacia Biotech) (28). Cells were stimulated
with GnRH agonists in the presence of 10 mM LiCl
for 1 h at 37 C with gentle agitation. Inositol phosphates were
extracted with 10 mM formic acid for a minimum of
30 min at 4 C (30) and separated on Dowex ion exchange columns. Total
inositol phosphates were eluted, and radioactivity was determined.
Data analysis
All assays were performed in duplicate and repeated three times.
Data analysis was performed using nonlinear regression, with sigmoidal
dose-response parameters (y = minimum response +
[(maximum response - minimum response)/(1 +
10(logEC50-x))], where y
represents the inositol phosphate production, and x
represents the log of the peptide concentration), using PRISM
(GraphPad, San Diego, CA). The peptide concentration required to
half-maximally stimulate inositol phosphate
(EC50) was calculated from the individual
sigmoidal dose-response curves and represents the
SEM of the three separate experiments (see Fig. 4D).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Sequencing of 5
different cDNA clones indicated 3 nucleotide differences among them. A
high fidelity polymerase was used in the PCR reactions; thus, these
differences were probably due to polymorphisms or to duplicate genes in
the tetraploid genome of X. laevis. Relevant regions of the
genomic clone were sequenced for confirmation. Theoretical translation
of the sequence revealed a 368-amino acid protein with 7 hydrophobic
TMs characteristic of a GPCR. This receptor has 46% and 55% amino
acid identity with the human and catfish GnRH receptors, respectively.
Figure 2 shows the deduced amino acid
sequence of the XLGnRH-R I aligned with the human, mouse, catfish, and
goldfish GnRH receptors (8, 10, 11).
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). The X.
laevis GnRH receptor gene, however, has an additional third intron
(intron I) at position -3 relative to the start ATG codon, which is
approximately 1.3 kb. Introns II and III are in the same positions as
the introns in the mouse and human GnRH receptor genes (31, 32). The
adaptor primer ligated at position -216 relative to the start codon.
Although this may indicate a transcription start site, it needs to
be confirmed by independent methods. The start ATG is situated in a
Kozac translation initiation consensus sequence (GCCATGG).
Receptor expression
Expression of X. laevis GnRH receptor messenger RNA
transcripts in total pituitary RNA could not be detected using Northern
blot (results not shown). The distribution of the receptor gene
expression was, therefore, investigated using RT-PCR. Expression of the
GnRH receptor could be detected in the pituitary and the midbrain
of adult X. laevis (Fig. 3A).
No detectable expression could be found in the forebrain, hindbrain,
sympathetic ganglia, testis, or peripheral tissues, including the
heart, kidney, and liver. ß-Actin signals were approximately equal in
all of these tissues (Fig. 3B).
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). This stimulation by
both mGnRH and cGnRH II could be completely inhibited by addition
of the mammalian GnRH receptor antagonist, antagonist 26
([Ac-D-p-ClPhe1,2,D-Trp3,D-Lys6,D-Ala10NH2]GnRH;
data not shown). Of the naturally occurring isoforms of GnRH
tested for the amphibian receptor, cGnRH II showed the highest potency
(EC50, 0.23 nM), followed
by salmon GnRH (sGnRH; EC50, 18
nM), mGnRH (EC50, 420
nM), and cGnRH I (EC50,
>10,000 nM; Fig. 4, A and D).
His5, Trp7, and
Tyr8 are required in combination for the high
activity of cGnRH II, as suggested by the decreased potencies of
[His5]GnRH and
[Trp7,Tyr8]GnRH (Fig. 4B).
125I-Labeled superactive agonists,
[D-Ala6,N-Me-Leu7,Pro9-NHEt]GnRH,
[His5,D-Arg6,Trp7,Tyr8]GnRH,
[His5,D-Tyr6]GnRH
(29), and antagonist 26 were used to investigate the binding affinity
of the X. laevis GnRH receptor. No binding could be detected
using either whole cell or membrane binding assays. The activities of
several other putative superactive GnRH agonists were therefore tested
to find a superactive agonist, which could be used to measure binding.
Buserelin, which has previously been shown to bind to the pituitary of
the green frog (33), had a lower potency than cGnRH II, whereas none of
the D-amino acid substitutions in position 6 of
cGnRH II
([His5,D-Arg6,Trp7,Tyr8]GnRH,
[His5,D-Trp6,Trp7,Tyr8]GnRH,
and
[His5,D-Lys6,Trp7,Tyr8]GnRH)
showed any increase in potency compared with cGnRH II (Fig. 4C). It is
unlikely, therefore, that any of these ligands would be suitable for
ligand binding studies.
We previously found poor binding of human (34) and goldfish (23) GnRH receptors expressed on COS-1 cells. Posttranslational modification of the transiently expressed receptor may differ from that of the endogenous X. laevis GnRH receptor and may cause low expression of the receptor, thus preventing the detection of binding. The X. laevis GnRH receptor, like the human GnRH receptor, has only one glycosylation consensus site in the amino terminus (Asn16). Incorporation of an additional glycosylation site in the human receptor, which occurs in the well expressed rat and mouse GnRH receptors, increased its expression. Binding studies on this human GnRH receptor construct confirmed the relative potencies of GnRH ligands from inositol phosphate studies (34).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Like the nonmammalian GnRH receptors from catfish and goldfish (22, 23), the amphibian receptor has an intracellular carboxyl-terminal tail that has been shown to be required for ligand-induced receptor desensitization and internalization (27, 36). Another feature common to nonmammalian GnRH receptors is the occurrence of aspartic acid residues in both TMs II and VII (Asp80 and Asp305, respectively), which are highly conserved as aspartic acid and asparagine, respectively, in GPCRs. The mammalian GnRH receptors have an asparagine in the corresponding position in TM II and an aspartic acid in TM VII (Asn87 and Asp318 in the human GnRH receptor). Mutation of Asn87 to Asp in the mammalian receptors, mimicking the nonmammalian GnRH receptors, led to an inactive receptor (37). The nonmammalian GnRH receptors must, therefore, have coordinated changes at other positions, enabling the receptor to accommodate the aspartic acid residues in both TMs. Mutations of the catfish GnRH receptor revealed that the aspartic acid residue in TM II (Asp90) is important for the functioning of the receptor (38).
The two cysteines that form a disulfide bridge between extracellular loops I and II, which have been shown for the mammalian GnRH receptor (39, 40) as well as other GPCRs (41), are conserved in nonmammalian GnRH receptors (Cys107 and Cys184 in XLGnRH-RI and Cys 114 and Cys196 in the human GnRH receptor). The cysteines, in the amino-terminus and extracellular loop II of the mammalian GnRH receptors (Cys14 and Cys200, respectively, of the human GnRH receptor), which have also been suggested to form a disulfide bridge (39), are not conserved in the XLGnRH-R I and the other cloned nonmammalian GnRH receptors. The complete absence of this bridge may affect the structural integrity of the receptors and hence contribute to the decreased selectivity for mGnRH seen in the nonmammalian receptors.
As expected of GPCRs that bind small peptide hormones (19), the amino-terminus is very poorly conserved among the mammalian, the X. laevis and the nonmammalian GnRH receptors. The TM domains important for ligand binding as well as the receptor conformation (41) are highly conserved. The amino acid residues in the TMs and the extracellular loops of the mammalian GnRH receptors, which have been shown to be important for ligand interactions, are conserved in XLGnRH-R I as well as in the nonmammalian GnRH receptors (18, 22, 23). This is, however, expected, as these residues are not responsible for GnRH selectivity because they are involved in binding to the amino- and carboxyl-termini of GnRH, which are conserved in all endogenous forms of GnRH (1, 2). These include Asp98 and Lys121 of the human GnRH receptor, which are both thought to interact with His2 of GnRH (Asp91 and Lys114 in XLGnRH-R I) (18, 42), and Asn102 at the extracellular surface of TM II, which has been shown to interact with the glycine amide of GnRH (Asn95 in XLGnRH-R I) (43). It is noteworthy, however, that Glu301 of the mouse GnRH receptor (or the equivalent Asp302 of the human GnRH receptor) in extracellular loop III, which is thought to be important for selectivity of Arg8 in mGnRH for the mammalian GnRH receptors (20), is conserved in the XLGnRH-R I (Glu288) and the other nonmammalian GnRH receptors despite their nonselectivity for this residue (15, 22, 23). However, unlike the goldfish GnRH receptor (23), XLGnRH-R I does show some discrimination for the Arg in position 8, as [Gln8]GnRH (cGnRH I) was at least 20-fold less potent than [Arg8]GnRH (mGnRH), which is similar to that observed for mammalian receptors. Nevertheless, it is clear that Glu288 of this receptor is not as powerful a determinant of ligand selectivity as in the mammalian receptor, because unlike in the mammalian receptors, cGnRH II and sGnRH, which lack Arg in position 8, are far more potent than mGnRH. Interestingly, a proline residue (Pro303) in the human GnRH receptor follows the crucial acidic residue, whereas a proline precedes the acidic Glu288 in the amphibian receptor. This amino acid change in the receptor may alter the conformation of the loop and thus change the orientation of the acidic residue side-chain and consequently the ligand selectivity. Exchange of the proline residue in the mammalian GnRH receptor to precede the acidic residue led to a decrease in distinguishing Arg8 (Fromme, B., unpublished results). The finding that all of the analogs with Trp in position 7 had an increased potency for XLGnRH-R I indicates that Trp7 may be a crucial determinant for binding to this receptor and similarly in the chicken GnRH receptor (21). Possibly there is a cognate interacting residue (or residues) in the X. laevis receptor that interacts with Trp in position 7.
The amino acid residues that have been shown to be important for G protein coupling and signal transduction are conserved among the mammalian GnRH receptors, XLGnRH-R I, and the other cloned nonmammalian GnRH receptors (22, 23), indicating that receptor activation may involve similar mechanisms in these receptors. These include the highly conserved DRY motif (DRH in the XLGnRH-R I), which is at the intracellular border of TM III. This motif and the conserved isoleucine residue that occurs two amino acid residues further downstream (Ile143 in the human GnRH receptor and Ile136 in XLGnRH-R I) have been found to be important in ligand-induced receptor activation and G protein coupling (44, 45). The NPX2–3Y motif, important for signal transduction, in the TM VII of GPCRs (DPLIY in the human GnRH receptor) (46) is conserved in the amphibian receptor (DPLVY in the XLGnRH-R I). Ala261 in intracellular loop III of the human GnRH receptor, important for G protein coupling and receptor internalization is also conserved (Ala247 in XLGnRH-R I) (47).
XLGnRH-R I is expressed in the pituitary and the midbrain of adult X. laevis. This receptor is, therefore, likely to play a role in the regulation of gonadotropin release from the pituitary. It is thus surprising that this receptor has a higher selectivity for cGnRH II compared with mGnRH, which is thought to be the central gonadotropin regulator in Amphibia. This unexpected characteristic, which conflicts with biological studies that showed that the amphibian pituitary was unselective for the different natural forms of GnRH (35), may be explained by three possibilities. Firstly, other subtypes of GnRH receptors may occur in Amphibia. Binding of GnRH to membranes from the sympathetic ganglia suggests the presence of a GnRH receptor in these ganglia (14). As for the pituitary GnRH receptor described here, cGnRH II shows a higher affinity than mGnRH for binding to sympathetic ganglia membranes (14). XLGnRH-R I expression was, however, not detected in the sympathetic ganglia or the hindbrain using RT-PCR. Thus, a second GnRH receptor subtype may exist in the sympathetic ganglia. The partial clone we have designated the type II GnRH receptor may represent this (15). Secondly, the up to 5-fold higher concentration of mGnRH in the preoptic and hypothalamic areas relative to cGnRH II (9) may compensate for the lower potency of mGnRH. Thirdly, this may be a typical characteristic of nonmammalian vertebrate GnRH receptors. The chicken, goldfish, and catfish GnRH receptors showed a much higher selectivity for cGnRH II (21, 22, 23). Studies of recombinant goldfish GnRH receptors also conflicted with biological studies of gonadotropin release in natural tissues (23).
The genomic structure of the X. laevis GnRH receptor differs from that in mammals. Although the X. laevis type I GnRH receptor has two introns at identical positions to the mammalian GnRH receptors (31, 32), there is an additional exon at position -3 relative to the start codon.
The unique structural and pharmacological features seen in the mammalian GnRH receptor must have, therefore, evolved after the divergence of mammals and Amphibia some 350–400 million yr ago. It is perhaps surprising to note that the nonmammalian features have been conserved between the Amphibia and bony fish (Osteichthyes) over such a long evolutionary period, as these phyla diverged even before the split of Amphibia and mammals. Certain selective physiological features that are unique to the mammalian reproductive system must have accelerated these unique receptor characteristics in mammals. One consequence of such a selective pressure is the lack of rapid ligand-induced receptor desensitization in the mammalian GnRH receptors, a phenomenon that may have evolved to facilitate the protracted LH surge required for ovulation and is presumably brought about by the absence of an intracellular carboxyl-terminal tail (48).
The characterization of the X. laevis GnRH receptor provides insights into the understanding of GnRH receptors in general, including ligand selectivity and receptor activation. Despite not being the expected intermediate between mammalian and nonmammalian vertebrates, it provides additional tools that can be exploited to further our understanding of receptor-ligand interactions in the GnRH system. For example, by the construction of chimeric receptors and using site-directed mutagenesis, amino acid residues could be identified that contribute to the altered ligand specificity of the X. laevis GnRH receptor compared with the mammalian GnRH receptors.
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Acknowledgments |
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Footnotes |
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Received July 30, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 R. Millar, S. Lowe, D. Conklin, A. Pawson, S. Maudsley, B. Troskie, T. Ott, M. Millar, G. Lincoln, R. Sellar, et al. A novel mammalian receptor for the evolutionarily conserved type II GnRH PNAS, August 1, 2001; (2001) 141048498. [Abstract] [Full Text] [PDF]
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), sGnRH
(
), mGnRH (
), and cGnRH I (
); B) cGnRH II (
