Complementary Deoxyribonucleic Acid Cloning, Gene ... - CiteSeerX

0013-7227/00/$03.00/0 Endocrinology Copyright © 2000 by The Endocrine Society

Vol. 141, No. 5 Printed in U.S.A.

Complementary Deoxyribonucleic Acid Cloning, Gene Expression, and Ligand Selectivity of a Novel Gonadotropin-Releasing Hormone Receptor Expressed in the Pituitary and Midbrain of Xenopus laevis* B. E. TROSKIE, J. P. HAPGOOD, R. P. MILLAR,

N. ILLING

AND

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 ABSTRACT We have cloned the full-length complementary DNA (cDNA) for a GnRH receptor from Xenopus laevis pituitary cDNA and determined its gene structure. The cDNA encodes a 368-amino acid protein that has a 46% amino acid identity to the human GnRH receptor. The X. laevis GnRH receptor has all of the amino acids identified in the mammalian GnRH receptors as sites of interaction with the GnRH ligand. However, this receptor cDNA shares the same distinguishing structural features of the GnRH receptor that have been characterized from other nonmammalian vertebrates. These include the pair of aspartate residues in the transmembrane domains II and VII compared with the aspartate/asparagine arrangement in mammalian receptors, the amino acid PEY motif in extracellular loop III (SEP in

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OST VERTEBRATE SPECIES contain two or more forms of GnRH. In general, the highly conserved chicken GnRH II (cGnRH II; [His5,Trp7,Tyr8]GnRH; designated type II GnRH) occurs along with one or more variant forms of GnRH (1, 2). Amphibians, like mammals, have the mammalian (mGnRH) and cGnRH II isoforms of GnRH (1– 5). mGnRH predominates in the preoptic area, hypothalamus, and median eminence and regulates the release of gonadotropins from the pituitary (6 –13). cGnRH II, which has a widespread distribution in the brain, but predominates in the hindbrain and spinal cord, including the sympathetic ganglia, may be important in neuromodulation (8 –14). The presence of two or more forms of GnRH suggests the expression of several cognate receptors. We previously reported the identification of two forms of GnRH receptor in Xenopus laevis from partial sequences encoding part of transmembrane domains VI and VII and extracellular loop III (15). These receptors were designated the type I or type II receptors based on the occurrence of conserved amino acid motifs in extracellular loop III (15). In this paper we report on Received July 30, 1999. 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: [email protected]. * The sequence of the X. laevis GnRH receptor has been submitted to GenBank (accession no. AF172330).

mammals), and the presence of a carboxyl-terminal tail. Previous studies have reported that mammalian GnRH was equipotent to other naturally occurring GnRH subtypes in stimulating LH release from the amphibian pituitary. However, in this study we show that the X. laevis GnRH receptor has ligand selectivity for the naturally occurring GnRHs similar to other nonmammalian GnRH receptors. The order of potency of the GnRHs in stimulating inositol phosphate production in COS-1 cells transiently transfected with the X. laevis GnRH receptor cDNA was chicken GnRH II⬎salmon GnRH⬎mammalian GnRH. Transcripts of this GnRH receptor are expressed in the pituitary and midbrain of X. laevis. (Endocrinology 141: 1764 –1771, 2000)

characterization of the full-length type I GnRH receptor complementary DNA (cDNA; XLGnRH-R I), that was cloned from cDNA prepared from pituitaries of adult X. laevis. 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

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THE X. laevis GnRH RECEPTOR

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 re-

FIG. 1. Schematic representation of the X. laevis genomic and cDNA clones showing the cloning strategy and the primers used for PCR amplifications. Clones generated from genomic DNA or cDNA are designated pGXL and pCXL, respectively. For details of the cloning strategy, see Materials and Methods.

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ceptors that also have mGnRH as their natural ligand in the pituitary. Materials and Methods GnRH analogs The following GnRH analogs were prepared by solid phase synthesis and purified by C18 reverse phase chromatography: mGnRH, [His5,Trp7,Tyr8]GnRH (cGnRH II), [Gln8]GnRH (cGnRH I), [His5]GnRH, [Trp7,Tyr8]GnRH,[His5,d-Tyr6]GnRH,[His5,d-Arg6,Trp7,Tyr8]GnRH,[His5, d-Trp6,Trp7,Tyr8]GnRH, [His5,d-Lys6,Trp7,Tyr8]GnRH, and [d-Ala6,NMe-Leu7,Pro9-NHEt]GnRH. [Ac-D-p-ClPhe1,2,d-Trp3,d-Lys6,d-Ala10NH2] GnRH (antagonist 26), and [Trp7,Leu8]GnRH (salmon GnRH) were gifts from D. Coy and R. W. Roeske, respectively.

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

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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). The pituitaries from 10 male frogs were pooled for RNA extraction. Total RNA was prepared by extraction with guanidium thiocyanate (24). cDNA was synthesized using the Marathon cDNA amplification kit (CLONTECH Laboratories, Inc., Palo Alto, CA). The cDNA was amplified in a thermal cycler (Perkin-Elmer Corp., Foster City, CA) in 50 ml containing 200 ␮m deoxy (d)-NTPs, adaptor primers, and the appropriate gene-specific primer (XL4a for the primary PCR and XL3a for the nested PCR) at 10 mm and 1 ⫻ Advantage KlenTaq polymerase mix (CLONTECH Laboratories, Inc.). PCR amplifications were performed as follows: denaturation at 94 C for 1 min, followed by 35 cycles of denaturation at 94 C for 30 sec, annealing at 64 C for 30 sec, and extension for 3 min at 68 C. Primary PCR products were diluted 1:500, for use in the secondary PCR. Products of all reactions were blotted and probed with the [␥-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 ␮g 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 ␮l water. The quality of the cDNA was assessed by running 2 ␮l 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 ␤-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 ␤-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 ⫻ 105 cells/well for inositol phosphate assays and 4 ⫻ 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 ␮g) 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

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(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).

Results The X. laevis pituitary GnRH receptor: cDNA and gene structure

The cloning strategy used to obtain the sequences of the 5⬘and 3⬘-untranslated regions of the X. laevis type I GnRH receptor (XLGnRH-R I) is summarized in Fig. 1. 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). XLGnRH-R I, like the human and mouse GnRH-Rs (31, 32), is encoded by three exons separated by two large introns (Fig. 1). 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


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FIG. 3. Expression of the X. laevis GnRH receptor cDNA (A) compared with the expression of ␤-actin cDNA (B), using PCR amplification of cDNA from the sympathetic ganglia, pituitary, midbrain, brain, forebrain, hindbrain, kidney, liver, heart, and testis. PCR was performed by 35 cycles of amplification, and products were blotted onto nitrocellulose and probed with the appropriate gene specific probes (see Materials and Methods). The negative control is where no cDNA template was added, whereas the positive control used either mouse ␤-actin (B) or the X. laevis GnRH receptor (pCXL5) (A) cDNA as template.

including the heart, kidney, and liver. ␤-Actin signals were approximately equal in all of these tissues (Fig. 3B). Biological activity of the amphibian GnRH receptor

FIG. 2. Amino acid sequence alignment of the cloned catfish (cfGnRH-R), goldfish A and goldfish B (gfGnRH-RA and gfGnRH-RB, respectively); X. laevis (xlGnRH-R) and the human (hGnRH-R) GnRH receptors, showing the transmembrane helix boundaries (18). Amino acid residues that are thought to be involved in glycosylation (⽧), disulfide bridge formation (*), interhelical interactions (), ligand binding (Œ), and G protein coupling (F) are indicated. The consensus sequence is included to show the amino acid residues, which are conserved between the nonmammalian and the human GnRH receptors.

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,

The recombinant X. laevis GnRH receptor transfected into COS-1 cells coupled to phospholipase C, as revealed by inositol phosphate production after stimulation with GnRH and various other GnRH agonists (Fig. 4). 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). 125 I-Labeled superactive agonists, [d-Ala6,N-Me-Leu7, 9 Pro -NHEt]GnRH, [His5,d-Arg6,Trp7,Tyr8]GnRH, [His5,dTyr6]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

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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). Discussion

FIG. 4. Shows the inositol phosphate production in COS-1 cells transfected with the X. laevis pituitary GnRH receptor (pCXL5) and stimulated with A) chicken GnRH II (f), sGnRH (Œ), mGnRH (⽧), and cGnRH I (); B) cGnRH II (f), [Trp7,Tyr8]GnRH (), [His5]GnRH (Œ), and [His5,D-Tyr6]GnRH (⽧); C) chicken GnRH II (f), buserelin (F), [His5,D-Trp6,Trp7,Tyr8]GnRH (), [His5,D-Arg6,Trp7,Tyr8]GnRH (Œ), and [His5,D-Lys6,Trp7,Tyr8]GnRH (⽧). The effective concentration for half-maximum stimulation (EC50) of inositol phosphate production by the peptides is shown (D). The values represent the SEM and were calculated from three separate experiments in which each dose was studied in duplicate.

GnRH receptors have been cloned and characterized in several mammalian (18) and nonmammalian vertebrate species (catfish, goldfish, and chicken; Sun, Y., unpublished results) (22, 23). The mammalian GnRH receptors have been shown to retain a high selectivity for mammalian GnRH compared with the other natural forms of GnRH, whereas the nonmammalian GnRH receptors have been shown to have a high selectivity for cGnRH II (21–23). These two groups of GnRH receptors have a number of major structural differences. Amphibia and mammals have both mGnRH and cGnRH II as endogenous GnRHs (1–13). In these animals, mGnRH is thought to be the central hypophysiotropic regulator, whereas cGnRH II, may have an additional role in neuromodulation (6 –14). Despite mGnRH being the prime regulator of gonadotropin release, the amphibian pituitary has previously been reported to be unselective for the different natural forms of GnRH (35). The amphibian GnRH pituitary receptor may, therefore, have been expected to represent an intermediate between the cloned nonmammalian GnRH receptors and mammalian GnRH receptors, as it binds to the same peptide (mGnRH) and has a different ligand selectivity, yet induces the same physiological response. Surprisingly, the GnRH receptor described in this study, XLGnRH-R I, has the typical structural features and pharmacological characteristics of the other cloned nonmammalian GnRH receptors (22, 23), including a particularly high selectivity for cGnRH II over mGnRH by approximately 2000-fold. 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 aminoterminus 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 carboxyltermini 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 hu-

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man 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 (21–23). Studies of recombinant gold-

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fish 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 carboxylterminal 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.

9. 10. 11.

12. 13.

14. 15. 16.

17. 18. 19. 20.

21.

22. 23.

Acknowledgment We thank Mr. J. Morta for technical assistance.

24.

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