Ala/Thr201 in Extracellular Loop 2 and Leu/Phe290 in Transmembrane Domain 6 of Type 1 Frog Gonadotropin-Releasing Hormone Receptor Confer Differential

http://www.100md.com 《内分泌学杂志》2003年第2期

     Abstract0o, http://www.100md.com

    Recently, we have identified three distinct types of bullfrog GnRH receptor (designated bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3). In the present study, we have isolated three GnRHR clones in Rana dybowskii (dyGnRHR-1, dyGnRHR-2, and dyGnRHR-3). Despite high homology of dyGnRHRs with the corresponding bfGnRHRs, dyGnRHRs revealed different signaling pathways and ligand sensitivity compared with the bfGnRHR counterparts. Activation of dyGnRHRs with GnRH stimulated cAMP-mediated gene expression. However, dyGnRHR-3 but not dyGnRHR-1 and -2 induced c-fos promoter-driven gene expression. Consistently, dyGnRHR-1 and dyGnRHR-2 were not able to increase GnRH-induced inositol phosphate accumulation, whereas all bfGnRHRs and dyGnRHR-3 were, indicating that dyGnRHR-1 and dyGnRHR-2 are coupled to solely G_s, whereas all bfGnRHRs and dyGnRHR-3 are coupled to both G_s and G_q/11. Moreover, dyGnRHR-1 and dyGnRHR-2 showed about 10-fold less sensitivity to each ligand than that of the bfGnRHR counterparts. Using type 1 chimeric and point-mutated receptors, we further elucidated that specific amino acids, Ala/Thr²⁰¹ in extracellular loop 2 and Leu/Phe²⁹⁰ in transmembrane domain 6 of the type 1 receptor, are responsible for ligand sensitivity and signal transduction pathway. Particularly, substitution of Leu²⁹⁰ to Phe in dyGnRHR-1 increased GnRH-induced inositol phosphate production as well as c-fos promoter-driven gene expression whereas substitution of Phe²⁹⁰ to Leu in bfGnRHR-1 decreased those activities. Collectively, these results demonstrate the presence of three types of GnRHR in amphibians, and suggest species- and type-specific ligand recognition and different signaling pathways in frog GnRHRs.

    Introductionua9, 百拇医药

    GnRH IS AN {alpha} -amidated decapeptide that plays a pivotal role in reproductive function in vertebrates (1). Following the elucidation of the primary structure of mammalian GnRH (mGnRH, pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂) (2, 3), a dozen isoforms of GnRH have been characterized in vertebrates. The primary structures of the different GnRH variants exhibit 70–90% sequence identity to mGnRH (4). Moreover, most vertebrate species contain two or more forms of GnRH in the brain (4). One form of GnRH is mammalian GnRH (mGnRH) (or structural variants of mGnRH), which predominates in the hypothalamus regulates the synthesis and secretion of gonadotropins in the pituitary. The other form is chicken GnRH-II (cGnRH-II; [His⁵, Trp⁷, Tyr⁸]GnRH) that was originally isolated from the chicken brain (5) but has now been identified in almost all vertebrate taxa including human (6). cGnRH-II neurons are mainly located in the midbrain and projects their nerve terminals to the posterior hypothalamus, hindbrain, and spinal cord (5, 6). Two GnRH variants, mGnRH and cGnRH-II, have been isolated from the brain of the European green frog Rana ridibunda (7), and we have recently cloned the cDNAs encoding two different forms of GnRH, mGnRH, and cGnRH-II, in the bullfrog R. catesbeiana (8), and [Trp⁸]GnRH, a mGnRH variant, in a Korean frog, R. dybowskii (9). A third form, salmon GnRH (sGnRH; [Trp⁷, Leu⁸]GnRH), is mainly found in the terminal nerve and olfactory system in fish (10, 11). The presence of a third form GnRH in amphibians and mammals is not yet established. However, a recent study using HPLC combined with RIA indicates the possible existence of an sGnRH-like peptide in the mammalian brain (12).

    The presence of two or more forms of GnRH suggests coevolution of their cognate receptors. Millar and his colleagues (13) have provided evidence for the presence of three putative GnRH receptor (GnRHR) subtypes from partial sequences encoding extracellular loop (ECL) 3 in many vertebrate genomes. Recently, we have cloned three distinct types of GnRHR in the bullfrog, designated bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3, respectively (14). These receptors exhibit different tissue distribution and ligand selectivity. Like mammalian (15, 16) and other nonmammalian GnRHRs (17, 18, 19), bfGnRHR-1 is predominantly expressed in the pituitary, indicating that this receptor is involved in the neuroendocrine control of gonadotrope activity. In contrast, bfGnRHR-2 and bfGnRHR-3 are expressed throughout the brain, suggesting that these receptors may mediate neurotransmission or neuromodulation by the second (and/or third) GnRH isoform in the brain. Interestingly, all three bfGnRHRs respond better to cGnRH-II than mGnRH. However, among the three receptors, bfGnRHR-2 exhibits the highest sensitivity to cGnRH-II, and bfGnRHR-3 has the highest sensitivity to sGnRH (14). All three receptors exhibit typical characteristics of nonmammalian GnRHRs (17, 18, 19). In contrast to mammalian GnRHRs, the three frog receptors contain an intracellular carboxyl tail that is known to be important for ligand-dependent receptor desensitization and internalization (20, 21). The Asn^2.50 residue that probably interacts with the Asp^7.49 residue in mammalian GnRHRs (22) is changed to Asp in bfGnRHRs. Such structural differences between mammalian and nonmammalian GnRHRs are likely to cause different cellular and physiological responses.

    In the present study, we have cloned three types of GnRHR in R. dybowskii, that are designated dyGnRHR-1, dyGnRHR-2, and dyGnRHR-3. Although dyGnRHRs showed a high degree of sequence similarity with their bfGnRHR counterparts, they exhibited different ligand sensitivities and recruited different signal transduction pathways. The elucidation of the structure-function relationship of nonmammalian GnRHRs is important not only for understanding the physiological consequence of GnRH-GnRHR interaction in the pituitary and brain, but also for developing GnRH agonists and antagonists that may have clinical applications in a wide range of reproductive disorders (23). Thus, we further investigated the structure-function relationships of bfGnRHRs and dyGnRHRs by using chimeric and point-mutated receptors. The data revealed that specific amino acids in ECL2 and transmembrane domain 6 (TM VI) appear to be important for ligand sensitivity and signal transduction coupling.\, 百拇医药

    Materials and Methods

    GnRHs70, 百拇医药

    mGnRH and the GnRH agonist (D-Ala⁶, N-Me-Leu⁷ GnRH) were purchased from Sigma(St. Louis, MO). cGnRH-II and sGnRH were purchased from American Peptide Co.(Sunnyvale, CA). [Trp⁸]GnRH and [His⁵, D-Tyr⁶]GnRH were synthesized in Peptron Research & Co. (Taejeon, Korea).70, 百拇医药

    Animals and tissue preparation70, 百拇医药

    R. dybowskii was collected from streams in the Chonnam area of South Korea and housed in flow-through tanks under simulated natural conditions. Frogs were killed by decapitation. The tissues of interest were quickly dissected, immediately frozen in liquid nitrogen, and stored at -80 C until use. Total RNA was extracted using Tri-reagent (Sigma) according to the manufacturers instructions. Poly (A)⁺ RNA was purified from total RNA using QIAGEN Oligotex mRNA kit (QIAGEN, Chatsworth, CA).70, 百拇医药

    Amplification of R. dybowskii GnRHR cDNAs by RT-PCR

    Fifty nanograms of pituitary or hindbrain poly (A)⁺ RNA were reverse transcribed using an oligo(deoxythymidine)_15–18 primer and SuperScript II Ribonuclease H^- reverse transcriptase (Life Technologies, Inc., Rockville, MD). These cDNAs served as templates for subsequent PCR amplification using two degenerate deoxyoligonucleotide primers, DG-F: 5'-GCMGCWTTMRTKCTRGTRGTKRTBAGC-3' and DG-R: 5'-GGTCATYTTYA-GSGTYYTYAKHCKDGC-3' (B=T to C or G, D=A or T or G, H=A or T or C, K=G or T, M=A or C, R=A or G, S=G or C, W=A or T, Y=T or C), which correspond to DNA sequences encoding conserved amino acid sequences in the TM III and VI of other GnRHRs. PCR conditions were as follows: denaturation at 95 C for 5 min, followed by 35 cycles at 94 C for 15 sec, at 55 C for 10 sec and at 72 C for 30 sec. PCR products of the expected size (~ 390 bp) were excised and purified using Geneclean II kit (Bio 101, La Jolla, CA). Next, the PCR products were subcloned into pGEM-T (Promega Corp., Madison, WI). Positive clones were isolated and purified using a QIAGEN plasmid Mini kit (QIAGEN). Plasmids containing an insert of the expected size were subjected to DNA sequence analysis by the dideoxy chain termination method (Sequenase, United States Biochemical Corp., Cleveland, OH) or by using the Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Foster City, CA) and an automated ABI prism 377 DNA sequencer (Perkin-Elmer). Because sequence analyses of each PCR fragment revealed very high homology with bfGnRHR counterparts, we directly used gene specific primers for amplifying cDNA encoding dyGnRHR open reading frame (14). Primers used in this study were as follows: dyGnRHR-1F (5'-CGCGAATTCGCCACCATGAATATCTCAAAGGAAGTTAGCAT-3'), dyGnRHR-1R (5'-CGCCTCTAGATTCATATTATACACATCTGTAATTGACT-3'), dyGnRHR-2 F (5'-CGCGAATTCGCCACCATGCAGCCAGCGATCGTAAATCGAAG-3'), dyGnRHR-2R (5'-CGCCTCTAGATTCAAAAGACAGACTGTACT GTGGTGGCC-3'), dyGnRHR-3 F (5'-CGCGAATTCGCCACCATGAACGCTAGTGACCAACCTATGG-3'), dyGnRHR-3R (5'-CGCCTCTAGATTCACATAAAGCTCTCTACGATTTGAC-3'). An EcoRI site (underlined) in the forward primers and the XbaI site (underlined) in the reverse primers were introduced. The Kozak translation initiation site is shown in italics in the forward primers. The coding region of each dyGnRHR cDNA was subcloned into the EcoRI and XbaI sites of the mammalian expression vector pcDNA3 (Invitrogen, San Diego, CA), generating pc-dyGnRHR-1, pc-dyGnRHR-2 and pc-dyGnRHR-3.

    Northern blot analysis;3}w\, http://www.100md.com

    Ten micrograms of total RNA isolated from pituitary, brain, liver, spleen, heart, testis, adrenal gland, and kidney of R. dybowskii, sampled in December, were electrophoresed in a 1% formaldehyde-agarose gel, and transferred by capillary action to a Zeta-Probe GT membrane (Bio-Rad Laboratories, Inc., Richmond, CA). Random primed dyGnRHR-1, dyGnRHR-2, and dyGnRHR-3 cDNA fragments were labeled (Roche Molecular Biochemicals) with [{alpha} -³²P] deoxy-CTP (Amersham Pharmacia Biotech, Buckinghamshire, UK) and used as probes for Northern blot analysis. Hybridization was performed as described elsewhere (14). The blots were washed twice with 2x SSC (1x SSC: 150 mM NaCl; 15 mM sodium citrate, pH 7.0), 0.1% sodium dodecyl sulfate at room temperature for 5 min each, and then once with 0.1 x SSC, 0.1% sodium dodecyl sulfate at 65 C for 50 min. The filters were then exposed to x-ray films (X-Omat, Kodak, Rochester, NY) at -70 C for about 1 wk.

    RT-PCR analysis of dyGnRHR mRNAs\#fpqou, http://www.100md.com

    The expression of dyGnRHRs in different tissues was analyzed by RT-PCR using total RNA, prepared from pituitary, forebrain (including olfactory lobe and telencephalon), midbrain/hindbrain (including mesencephalon, hypothalamus, cerebellum, and spinal cord), testis, and adrenal gland of R. dybowskii (sampled in December). RT-PCR conditions were as follows: first strand synthesis from 5 µg/20 µl of each total RNA was primed with oligo-deoxythymidine_15–18 primers and reverse-transcribed with SuperScript II Ribonuclease H^- reverse transcriptase (Life Technologies, Inc.) using the manufacturer’s protocol. Then, 0.2 µl of the first strand cDNA was amplified by PCR in a 10 µl volume using 2 mM deoxynucleotide triphosphates, 10 mM of primer, and the Advantage cDNA polymerase (CLONTECH Laboratories, Inc., Palo Alto, CA). The dyGnRHR-1 PCR was performed with primers dy1-F (5'-CCAAGCGCATGAGCAAAGGAACACTTTC-3') and dyGnRHR-1R (see above). The dyGnRHR-2 PCR was performed with primers dyGnRHR-2 F and dyGnRHR-2R, as described above. The dyGnRHR-3 PCR was performed with primers dy3-F (5'-TGTTTGTGTTCCACACGGTGAGCCGGTC-3') and dyGnRHR-3R (see above). The PCR cycling parameters were denaturation at 95 C for 30 sec, followed by 30 cycles at 94 C for 15 sec, 50 C for 10 sec, and 72 C for 30 sec. The whole PCR was electrophoresed on a 1.2% agarose gel, stained with ethidium bromide, and photographed under a UV light source. The different size products were then transferred to nylon membrane and hybridized with radiolabeled dyGnRHR cDNAs. As an internal control, the R. dybowskii glyceraldehyde-3-phosphate dehydrogenase (GAPDH) specific primers GAPDH-F (5'-CAATCCAATGGGGAGCTTCTG-3') and GAPDH-R (5'-CAATCCAATGGGGAGC-TTCTG-3') were used.

    Chimeric and point-mutated frog GnRHR-1+2, http://www.100md.com

    The cDNAs of bfGnRHR-1 and dyGnRHR-1 possess the HindIII and BamHI sites in TM III and TM V, respectively. We cleaved these sites to produce three fragments (N-terminal, middle, C-terminal part) and then ligated these fragments to generate three chimeric receptors. The bfGnRHR-1 fragment cut by EcoRI and BamHI was inserted in the EcoRI- and BamHI-cut pc-dyGnRHR-1 vector, generating bfBdy. The bfGnRHR-1 fragment cleaved by EcoRI and HindIII was inserted in the EcoRI- and HindIII-cut pc-dyGnRHR-1 vector, producing bfHdy. The dyGnRHR-1 fragment cleaved by EcoRI and BamHI was inserted in the EcoRI- and BamHI-cut pc-bfGnRHR-1 vector, generating dyBbf. Each point-mutated dyGnRHR-1 was generated by PCR-base mutagenesis. Each A²⁰¹, K²¹¹, T²⁶⁹, L²⁹⁰, and F²⁹⁹ residue of dyGnRHR-1 was changed to that of bfGnRHR-1, producing A²⁰¹T, K²¹¹E, T²⁶⁹M, L²⁹⁰F, and F²⁹⁹Y mutant, respectively. Double amino acid substitution of A²⁰¹ and L²⁹⁰ to T and F was made, designated A²⁰¹TL²⁹⁰F. Each M²⁶⁹, F²⁹⁰, and Y²⁹⁹ residue of bfGnRHR-1 was changed to that of dyGnRHR-1, producing M²⁶⁹T, F²⁹⁰L, and Y²⁹⁹F mutants, respectively. Double amino acid substitution of F²⁹⁰ and Y²⁹⁹ to L and F, respectively, was designated F²⁹⁰LY²⁹⁹F.

    Cell culture, transient transfection, and luciferase (luc) assaysi+, http://www.100md.com

    HeLa or CV-1 cells were maintained at 37 C in DMEM with 10% heat-inactivated fetal bovine serum, 1 mM glutamate, 100 U of penicillin, and 100 µg/ml streptomycin. Cells were cultured in 24-well plates and transfection was performed using the SuperFect transfection kit (QIAGEN) according to the manufacturer’s instructions. For each transfection, 100 ng of each receptor cDNA, and 200 ng of pCRE-luc [containing four copies of the cAMP response element (CRE)], TGACGTCA, (Stratagene, La Jolla, CA) or c-fos-luc (containing -711 ~ +45 sequence of human c-fos promoter constructed in the pFLASH vector, a gift from Dr. R. Prywes, Columbia University, New York, NY) along with 200 ng of internal control plasmid pCMVßGal were used. The empty vector pcDNA3 was used to adjust the total amount of DNA transfected to 0.7 µg. About 24 h after transfection, cells were treated with mGnRH, cGnRH-II, or [Trp⁸]GnRH. For c-fos-luc analysis, cells were maintained in serum-free DMEM for 24 h before treatment with GnRH. Six hours after GnRH treatment, cells were harvested, and the luc activity in the cell extract was determined using a luc assay system according to standard methods in a Lumat LB9501 (EG & G Berthold, Bad Wildbad, Germany). The luc activities were normalized using the ß-galactosidase values. Transfection experiments were performed in duplicate and repeated three to five times. All data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA and, where P < 0.05, followed by the Bonferroni test. A P < 0.05 was considered to be significant.

    Ligand binding assayhk*gl, 百拇医药

    cGnRH-II and [His⁵, D-Tyr⁶]GnRH were radioiodiated using the chloramine-T method. Cell membranes were prepared 48 h after transfection. Membranes resuspended in protein-free binding buffer (40 mM Tris, pH 7.4; 2 mM MgCl₂). The membrane fraction (20 µg protein per tube) was incubated with I¹²⁵-cGnRH-II or I¹²⁵-[His⁵, D-Tyr⁶]GnRH with a range of concentrations (0.1–4 nM) and 0.1% BSA. The membrane preparations were incubated with tracer on ice overnight. Nonspecific binding was determined by adding 100 µM cold cGnRH-II or [His⁵, D-Tyr⁶]GnRH. For displacement experiments membranes were incubated on ice overnight with I¹²⁵-cGnRH-II or I¹²⁵-[His⁵,D-Tyr⁶]GnRH (100,000 cpm), 0.1% BSA, and varying concentrations of mGnRH, cGnRH-II, [Trp⁸]GnRH, and [His⁵, D-Tyr⁶]GnRH. The reaction was terminated by filtration through GF/C filter (Brandel, Inc., Gaithersburg, MD) that were presoaked with binding buffer containing 1% BSA, and washed twice with binding buffer.

    Inositol phosphate (IP) assayte, 百拇医药

    Twenty-four hours before transfection, CV-1 cells (1 x 10⁵ per well) were plated out in 12-well plates. The next day, the cells were transfected using SuperFect (QIAGEN). After transfection, the cells were incubated in inositol-free DMEM (Life Technologies, Inc.) containing 2% dialyzed fetal bovine serum and labeled with 1 µCi myo-[³H]inositol/well (Amersham Pharmacia Biotech) for 20 h. Medium was then removed and cells were washed with 0.5 ml buffer A (140 mM NaCl, 20 mM HEPES, 4 mM KCl, 8 mM D-glucose, 1 mM MgCl₂, 1 mM CaCl₂, 1 mg/ml fatty acid-free BSA). Then cells were preincubated with buffer A containing 10 mM LiCl for 15 min, followed by addition of graded concentration of the GnRHs at 37 C for 30 min. The reaction was stopped by removing the incubation medium and adding 0.5 ml ice-cold 10 mM formic acid. After 30 min at 4 C, the formic acid extracts were transferred into columns containing Dowex anion exchange resin. Total IPs were then eluted with 1 ml of 1 M ammonium formate/0.1 M formic acid, and the radioactivity was determined. The GnRH concentrations inducing half-maximal stimulation (EC₅₀) were calculated using the GraphPad Software, Inc. PRISM2 software.

    Results.r5p8e*, http://www.100md.com

    Identification of three types of GnRHR in R. dybowskii.r5p8e*, http://www.100md.com

    We have recently identified three types of GnRHR in the bullfrog (14). In this study, we attempted to isolate the cDNAs encoding orthologous GnRHRs in R. dybowskii. PCR experiments with degenerated primers, DG-F and DG-R that were used in a previous study (14), led us to amplify three putative cDNAs with strikingly high homology to the bfGnRHR cDNAs. Thus, we directly amplified full-length cDNAs encoding dyGnRHRs using bullfrog gene specific primers. Sequence analysis of the open reading frame of each dyGnRHR cDNA revealed a high degree of sequence identity (96–97%) with the bfGnRHR counterparts. Deduced amino acid sequences for the three open reading frame cDNAs revealed that type 1, type 2, and type 3 receptors encode 407-, 371-, and 424-amino acid proteins, respectively. Like other G protein-coupled receptors, dyGnRHRs possess seven putative transmembrane domains, two or three potential glycosylation sites in the N-terminal domain (at positions 2, 27, and 32 for dyGnRHR-1; 9 and 19 for dyGnRHR-2; 2, 25, and 41 for dyGnRHR-3), and several phosphorylation sites in intracellular loops 2 and 3 (Fig. 1) Like other nonmammalian GnRHRs, dyGnRHRs contain an intracellular carboxyl-terminal tail consisting of 74 (dyGnRHR-1), 57 (dyGnRHR-2), and 79 (dyGnRHR-3) residues, respectively. Residues, Asp⁹⁸, Asn¹⁰², and Lys¹²¹, which have been shown to play a crucial role in ligand binding in the human GnRHR, are conserved in homologous positions in the three dyGnRHRs. Each type of dyGnRHR exhibited striking homology with the corresponding bfGnRHR in both nucleotide and predicted amino acid sequences. Indeed, dyGnRHR-1 and dyGnRHR-2 share 96% amino acid identity with bfGnRHR-1 and bfGnRHR-2, and dyGnRHR-3 shares 97% amino acid identity with bfGnRHR-3. Compared with mammalian GnRHRs, dyGnRHR-1 and dyGnRHR-3 exhibited 31–33% amino acid identity, whereas dyGnRHR-2 exhibited 35–36% amino acid identity (Table 1). When compared with other nonmammalian GnRHRs, dyGnRHR-1 and dyGnRHR-3 exhibited 39–42% amino acid identity, whereas dyGnRHR-2 exhibited 48–50% amino acid identity (Table 1). Interestingly, dyGnRHR-3 revealed 85% sequence similarity with the Xenopus GnRHR (19). It is also of interest to note that dyGnRHR-3 showed a relatively high degree of sequence similarity (48%) with monkey GnRHR-2 (24, 25). In contrast, the different dyGnRHRs only shared modest sequence identity with each other. dyGnRHR-1 exhibited 40% amino acid identity with dyGnRHR-2, and 52% with dyGnRHR-3, whereas dyGnRHR-2 exhibited 39% identity with dyGnRHR-3.

    fig.ommitteed^#3, 百拇医药

    Figure 1. Sequence alignment of dyGnRHRs and bfGnRHRs. Amino acid sequences of the indicated GnRHRs were aligned using a MacVector software. The gray shaded regions indicate conserved amino acids, and the black shaded regions indicate the amino acid substitutions between the bullfrog and R. dybowskii orthologous receptor. The numbers on the right indicate respective amino acid positions. The putative transmembrane (TM) domains are indicated above the aligned sequences.^#3, 百拇医药

    fig.ommitteed^#3, 百拇医药

    Table 1. Amino acid identity among GnRHRs^#3, 百拇医药

    Differential expression of the three dyGnRHR types^#3, 百拇医药

    Northern blot analysis of total RNA was performed to determine the tissue distribution of dyGnRHRs. The dyGnRHR-1 probe revealed the existence of two molecular variants of transcript (2.7 and 3.2 kb) in the pituitary and only a weak hybridization signal (2.7 kb) in the brain (Fig. 2). The dyGnRHR-2 probe produced an intense signal (9.5 kb) in the brain only (Fig. 2). The dyGnRHR-3 probe revealed a single band (~ 7.5 kb) in the brain and a faint signal in the pituitary. The dyGnRHR-3 probe also hybridized with a different RNA variant (4.5 kb) in the liver (data not shown). The tissue distribution of the three types of dyGnRHR was further investigated by RT-PCR. The cDNA for dyGnRHR-1 was strongly amplified in the pituitary and hindbrain, whereas weak signals were seen in the forebrain, testis, and adrenal gland. The cDNAs for dyGnRHR-2 and dyGnRHR-3 were abundant in the hindbrain whereas faint signals were observed in the forebrain, testis, adrenal gland, and pituitary (data not shown).

    fig.ommitteed(}hy[(, 百拇医药

    Figure 2. Differential expression of three R. dybowskii GnRHRs. Total RNA (10 µg) prepared from pituitary (P) and brain (B) of R. dybowskii sampled in December were subjected to Northern blot analysis under high stringency conditions. The sizes of the different mRNA transcripts are indicated on the right. dyGnRHR-1 mRNA was primarily detected in the pituitary but faintly in the brain. dyGnRHR-2 mRNA was exclusively detected in the brain. dyGnRHR-3 mRNA was expressed strongly in the brain but weakly in the pituitary. 28S and 18S ribosomal RNAs that were applied to Northern blot were seen in the bottom.(}hy[(, 百拇医药

    Ligand binding assay of dyGnRHRs and bfGnRHRs(}hy[(, 百拇医药

    The biochemical characteristics of each GnRHR were studied on transfected HeLa cells by using I¹²⁵-cGnRH-II as a radioligand (Table 2). Each dyGnRHR revealed a similar ligand affinity and expression level when compared with its bfGnRHR counterpart. However, there were remarkable differences in ligand affinity and expression level among the three types of frog GnRHR. In particular, type 1 and type 2 receptors showed a relatively higher affinity to cGnRH-II than type 3 receptor as revealed by dissociation constant values. Interestingly, the membrane expression level of type 3 receptor was about 20 times higher than that of type 1 and type 2 receptors (Table 2).

    fig.ommitteed\?, 百拇医药

    Table 2. Ligan selectivity of bfGnRHRs and dyGnRHRs\?, 百拇医药

    Species-specific signal transduction coupling of dyGnRHRs and bfGnRHRs\?, 百拇医药

    To investigate the signaling pathways associated with the different GnRHRs, HeLa and CV-1 cells were transfected with plasmid containing each GnRHR and the CRE-luc (or c-fos-luc) reporter vector. In both HeLa and CV-1 cells, forskolin (FKN, 10 µM), an adenylate cylase activator, but not 12-O-tetradecanoylphenol-13-acetate (TPA, 200 mM), a protein kinase C activator, substantially increased luc activity in CRE-luc-cotransfected cells, whereas TPA but not FKN increased luc activity in c-fos-luc-cotranfected cells. Moreover, FKN-induced CRE-luc activity was completely inhibited by H89 (10 µM), a specific PKA inhibitor, but partially inhibited by GF109203X (5 µM), a specific protein kinase C inhibitor. TPA-induced c-fos-luc activity was completely blocked by GF109203X but partially by H89 (data not shown). The partial inhibition of either FKN-induced CRE-luc activity by GF109203X or TPA-induced c-fos-luc activity by H89 is most likely due to the partial cross-reactivity of these chemicals. These data indicate that cotranfection of GnRHR-containing plasmids with CRE-luc or c-fos-luc makes it possible to distinguish GnRHR-mediated signal transduction pathways. In CRE-luc-cotransfected cells, cGnRH-II significantly increased luc activity in all cells harboring each GnRHR with some variations in fold-increase. The highest fold induction was observed in bfGnRHR-2-transfected cells. dyGnRHR-1 and dyGnRHR-2 were less capable of inducing CRE-luc activity when compared with the orthologous bfGnRHRs, whereas dyGnRHR-3 exhibited similar activity to induce CRE-luc activity as bfGnRHR-3. Considering that dyGnRHR-1 and dyGnRHR-2 had similar ligand affinity and expression levels as their bfGnRHR counterparts (Table 2), it is plausible that dyGnRHR-1 and dyGnRHR-2 have less coupling efficiency than their bfGnRHR orthologs. Interestingly, in c-fos-luc-cotransfected cells, dyGnRHR-1 and dyGnRHR-2 did not increase luc activity whereas dyGnRHR-3 and all bfGnRHRs substantially increased luc activity (Fig. 3). This observation indicates that dyGnRHR-1 and dyGnRHR-2 preferentially couple to a G_s but marginally to a G_q/11, whereas all three bfGnRHRs and dyGnRHR-3 couple to both G_s and G_q/11. To ascertain whether dyGnRHR-1 and dyGnRHR-2 do not couple to the G_q/11, GnRH-induced IP accumulation was determined in cells bearing each GnRHR. In consistency, cGnRH-II induced a dose-dependent increase in IP production in cells expressing bfGnRHRs and dyGnRHR-3, but not in those expressing dyGnRHR-1 and dyGnRHR-2 (Fig. 4 A–C). To examine the ligand specificity for GnRH-induced IP accumulation, a variety of GnRH isoforms were tested. All GnRH ligands used failed to increase IP formation in cells expressing dyGnRHR-1 or dyGnRHR-2, whereas all ligands substantially augmented IP accumulation in cells harboring dyGnRHR-3 (Fig. 4D). This result again confirms that dyGnRHR-1 and dyGnRHR-2 marginally couple to G_q/11.

    fig.ommitteed9}y()*|, http://www.100md.com

    Figure 3. Differential signal transduction pathways of dyGnRHRs and bfGnRHRs. Each GnRHR expression vector was cotransfected with the CRE-luc (A) or c-fos-luc (B) reporter into HeLa cells. To determine the c-fos-luc activity, cells were maintained in serum-free medium for 24 h before GnRH treatment. Six hours after treatment with FKN (10 µM), TPA (200 nM), or cGnRH-II (1 µM), luc activities were determined. Note that CRE-luc activity was increased by FKN but not by TPA, whereas c-fos-luc activity was augmented by TPA but not by FKN, indicating that these reporter systems are useful tools for identifying specific signal transduction pathways. The experiments were repeated at least three times. The results represent the means ± SEM of three independent experiments.9}y()*|, http://www.100md.com

    fig.ommitteed9}y()*|, http://www.100md.com

    Figure 4. IP accumulation in cells expressing dyGnRHR or bfGnRHR in response to GnRH. A–C, CV-1 cells (1 x 10⁵ per well) were transfected with each GnRHR-containing plasmid. One day after transfection, the cells were incubated in inositol-free DMEM containing 2% dialyzed fetal calf serum and labeled with 1 µCi myo-[³H]inositol/well for 20 h. Medium was then removed and cells were preincubated with buffer A containing 10 mM LiCl for 15 min, followed by addition of the GnRHs at various concentrations at 37 C for 30 min. Total IP accumulation was assayed as described in Materials and Methods. Concentration-dependent stimulation of IP production in cells expressing each type of GnRHR was observed. D, Cells expressing each type of dyGnRHRs were exposed to various GnRHs (100 nM). Bars represent the mean ± SEM of % IP accumulation over the control group from three independent experiments.

    Species specificity in ligand sensitivity of dyGnRHRs and bfGnRHRs6ml$, 百拇医药

    We examined the ligand sensitivity using the CRE-luc reporter system with three natural GnRH ligands, i.e. cGnRH-II, mGnRH, and [Trp⁸]GnRH. dyGnRHR or bfGnRHR cDNA was cotransfected with the CRE-luc reporter vector into HeLa cells. For bfGnRHR-1 and dyGnRHR-1, three GnRH peptides exhibited a similar potency to activate receptors in terms of EC₅₀ value but dyGnRHR-1 was about 10 times less sensitive to the ligands than bfGnRHR-1 (Fig. 5 and Table 2). Differences in ligand sensitivity between bfGnRHR-1 and dyGnRHR-1 will be discussed in more detail hereafter (see chimeric receptor experiments). For bfGnRHR-2 and dyGnRHR-2, cGnRH-II exhibited a higher potency to activate receptors than mGnRH and [Trp⁸]GnRH. bfGnRHR-2 showed approximately 4-fold higher sensitivity to cGnRH-II than dyGnRHR-2. Moreover, dyGnRHR-2 marginally responded to [Trp⁸]GnRH and mGnRH. Overall, there were remarkable decreases in ligand sensitivity of dyGnRHR-2 compared with bfGnRHR-2. For bfGnRHR-3 and dyGnRHR-3, cGnRH-II came first and [Trp⁸]GnRH came second in terms of sensitivity (Table 2). It is of interest to note that [Trp⁸]GnRH exhibited a higher potency to activate all frog GnRHRs than mGnRH. This observation indicates that [Trp⁸]GnRH, a natural ligand for R. dybowskii, is evolutionarily adapted to frog GnRHRs.

    fig.ommitteed5of{!, 百拇医药

    Figure 5. Comparison of ligand sensitivity between dyGnRHRs and bfGnRHRs. HeLa cells were cotransfected with the CRE-luc reporter and GnRHR cDNAs. After a 48-h incubation, cells were treated with graded concentrations of different GnRHs for 6 h and luc activity was determined. The transfection was performed in duplicate and the results represent means ± SEM of three independent experiments.5of{!, 百拇医药

    Analysis of type 1 chimeric GnRHRs5of{!, 百拇医药

    Although bfGnRHR-1 and dyGnRHR-1 are clearly orthologous (their sequences differ by 13 amino acids), they exhibited different ligand sensitivity and signal transduction pathways. Thus, we further analyzed the structure-function relationship of bfGnRHR-1 and dyGnRHR-1 by constructing chimeric receptors. The cDNAs encoding the two receptors both possess HindIII and BamHI sites in TM III and TM V, respectively (Fig. 6A). We cleaved these sites to produce three fragments (N-terminal, middle, C-terminal part) and ligated these fragments to generate three chimeric receptors, i.e. bfBdy, bfHdy, and dyBbf (Fig. 6B). The ligand sensitivity of these chimeric receptors was examined in CRE-luc-cotransfected cells. cGnRH-II increased luc activity in cells expressing wild-type and all chimeric receptors in a dose-dependent manner. In terms of sensitivity to the ligand, EC₅₀ values (Fig. 6B) revealed that the bfBdy receptor behaved like wild-type bfGnRHR-1 whereas bfHdy receptor behaved like dyGnRHR-1. The sensitivity of the dyBbf receptor was comprised between those of bfGnRHR-1 and dyGnRHR-1. This result indicates that the middle part, especially two amino acids, Ala/Thr²⁰¹ and Lys/Glu²¹¹, in ECL2 of type 1 receptor appears important for ligand sensitivity. In terms of maximum response to ligand obtained by treatment of cells with a high concentration of cGnRH-II (100 nM), the dyBbf receptor behaved like bfGnRHR-1 whereas the bfHdy receptor behaved like dyGnRHR-1 (Fig. 6C). The maximum response of the bfBdy receptor resided between those of bfGnRHR-1 and dyGnRHR-1. It is important to note that, among the chimeric receptors, only the dyBbf receptor increased IP accumulation in response to GnRH stimulation and revealed an increase in ligand sensitivity (Fig. 6, D and E). This result indicates that the C-terminal region of the type 1 receptor plays a crucial role in IP production.

    fig.ommitteed%{;w|, http://www.100md.com

    Figure 6. Functional characterization of chimeric type 1 frog GnRHRs. A, Schematic diagram of dyGnRHR-1 and bfGnRHR-1. Shaded boxes with roman numerals indicate respective transmembrane domains. The amino acid residues that differ between dyGnRHR-1 and bfGnRHR-1 are indicated above. HindIII site at TM III and BamHI sites at TM V are seen. B, Schematic representation of the various chimeric receptors. The chimeric receptors were created by domain swapping through the common HindIII, and BamHI sites. EC₅₀ values obtained from GnRH-induced CRE-luc activity are indicated on the right side. C, HeLa cells were cotransfected with 200 ng of CRE-luc and indicated wild-type dyGnRHR-1 (dy1), bfGnRHR-1 (bf1), or chimeric GnRHRs. Forty-eight hours after transfection, cells were treated with 100 nM cGnRH-II for 6 h and luc activity was measured. D, CV-1 cells were transfected with indicated wild-type dy1, bf1, or chimeric GnRHRs and maintained in inositol-free medium containing 1 µCi myo-[³H]inositol for 20 h. Transfected cells were exposed to graded concentrations of cGnRH-II for 30 min and the amount of newly formed total IP was determined. E, IP formation was examined in cells expressing each chimeric receptor in response to 100 nM cGnRH-II. Data represent means ± SEM of three independent experiments.

    Analysis of point-mutated type 1 GnRHR[d)8(c, 百拇医药

    Because the chimeric receptor study indicated that the middle and C-terminal portion of the type 1 receptor are responsible for differential ligand sensitivity and signal transduction pathways, we mutated amino acids of dyGnRHR-1 to those of bfGnRHR-1. Using the CRE-luc assay, we found that the A²⁰¹T, T²⁶⁹M, L²⁹⁰F, and A²⁰¹TL²⁹⁰F mutant receptors increased luc activity like wild-type dyGnRHR-1, whereas luc activity by the K²¹¹E and F²⁹⁹Y mutants were decreased when compared with wild-type dyGnRHR-1 (Fig. 7A). Interestingly, the EC₅₀ values for A²⁰¹T, L²⁹⁰F and A²⁰¹TL²⁹⁰F mutants were lower than that of bfGnRHR-1 whereas the EC₅₀ values for K²¹¹E and T²⁶⁹M receptors were higher than that of dyGnRHR-1 (Fig. 7B). This result indicates that Thr²⁰¹ in the ECL2 and Phe²⁹⁰ in TM VI of bfGnRHR-1 positively affect ligand sensitivity, whereas Glu²¹¹ in ECL2 and Met²⁶⁹ in ICL3 negatively affect ligand sensitivity. This result is consistent with the chimeric receptor study in which increases in ligand sensitivities were observed in the bfBdy and dyBbf receptors comparing with wild-type dyGnRHR-1. Regarding G_q/11-mediated signal transduction, Leu/Phe²⁹⁰ in TM VI appears important because L²⁹⁰F mutant remarkably increased c-fos promoter-driven gene expression and IP formation in response to GnRH (Fig. 7, C and D). A slight increase in c-fos-luc activity was observed in the A²⁰¹T mutant but no significant increase in IP formation of A²⁰¹T mutant was detected. This difference is most likely due to different sensitivity of assay systems. A slight changes in IP production that was not detected in the IP assay system, can be detected in c-fos-luc assay system through an amplification of a signaling cascade, allowing us to observe significant increase in c-fos-luc activity in cells expressing A²⁰¹T mutant as shown in Fig. 7C. The K²¹¹E, T²⁶⁹M, and F²⁹⁹Y mutants were not able to increase either GnRH-induced c-fos-luc-mediated gene expression or IP accumulation. Together with the chimeric receptor study in which only the dyBbf receptor exhibited GnRH-induced IP accumulation, this result indicates that Leu/Phe²⁹⁰ in TM VI of the type 1 receptor is critical for G_q/11-mediated signal transduction. To further confirm this issue, Met²⁶⁹, Phe²⁹⁰, and Tyr²⁹⁹ residues of bfGnRHR-1 were mutated to Thr, Leu, and Phe, respectively. The F²⁹⁰L and Y²⁹⁹F mutants significantly reduced c-fos-driven gene expression and IP production compared with wild-type bfGnRHR-1. A double mutant, F²⁹⁰LY²⁹⁹F completely lost the ability to induce c-fos-luc activity and IP production (Fig. 8). However, the M²⁶⁹T mutant did not affect ability to induce G_q/11-mediated signaling pathway. This result again corroborates that Leu/Phe²⁹⁰ in TM VI is responsible for G_q/11-mediated signal transduction.

    fig.ommitteed-$0^, http://www.100md.com

    Figure 7. Functional characterization of point-mutated dyGnRHR-1. A-C, HeLa cells were transfected with the CRE-luc (A and B) or c-fos-luc (C) reporter and point-mutated GnRHR. Each A²⁰¹, K²¹¹, T²⁶⁹, L²⁹⁰, and F²⁹⁹ residue of dyGnRHR-1 was changed to that of bfGnRHR-1, producing A²⁰¹T, K²¹¹E, T²⁶⁹M, L²⁹⁰F, and F²⁹⁹Y mutants, respectively. A double mutant, A²⁰¹TL²⁹⁰F, was also made. Forty-eight hours after transfection, cells were treated with 100 nM cGnRH-II for 6 h and luc activity was measured. EC₅₀ values were obtained from GnRH-induced CRE-luc activity (B). D, CV-1 cells were transfected with the wild-type or point-mutated GnRHR and maintained in inositol-free medium containing 1 µCi myo-[³H]inositol for 20 h. Total IPs were eluted, and the radioactivity was determined. Data represent means ± SEM of three independent experiments.-$0^, http://www.100md.com

    fig.ommitteed-$0^, http://www.100md.com

    Figure 8. Functional characterization of point-mutated bfGnRHR-1. A, CV-1 cells were transfected with c-fos-luc reporter and mutated bfGnRHRs. Each M²⁶⁹, F²⁹⁰, and Y²⁹⁹ residue of bfGnRHR-1 was changed to that of dyGnRHR-1, producing M²⁶⁹T, F²⁹⁰L, and Y²⁹⁹F mutants, respectively. Double amino acid substitutions of F²⁹⁰ and Y²⁹⁹ to L and F, respectively, was designated F²⁹⁰LY²⁹⁹F. Forty-eight hours after transfection, cells were treated with 100 nM cGnRH-II for 6 h and luc activity was measured. B, CV-1 cells were transfected with wild-type or mutated bfGnRHR and maintained in inositol-free medium containing 1 µCi myo-[³H]inositol for 20 h. Total IPs were eluted and the radioactivity was determined. Data represent means ± SEM of three independent experiments.

    Discussion7, http://www.100md.com

    The present study has demonstrated the presence of three types of GnRHR in R. dybowskii as previously reported in the bullfrog (14). Interestingly, dyGnRHRs exhibited marked differences in ligand recognition and signal transduction compared with the orthologous bfGnRHR even though they shared over 96% homology in amino acid sequences. Studies on structure-function relationships of the type 1 receptor have shown that Ala/Thr²⁰¹ in ECL2 and Leu/Phe²⁹⁰ in TM VI are critical for differential ligand sensitivity and signal transduction of type 1 frog GnRHR.7, http://www.100md.com

    The identification of at least three variants of GnRH in the brain of a single species (12) suggested the existence of multiple receptor isoforms (13, 14). Because cell bodies expressing cGnRH-II are localized in the midbrain and because their nerve terminals are distributed in the central nervous system, the receptors for cGnRH-II is expected to be present in the brain. In agreement with previous studies conducted in the bullfrog (14), we found that dyGnRHR-2 and dyGnRHR-3 but not dyGnRHR-1 are primarily expressed in the brain, whereas dyGnRHR-1 is mainly expressed in the pituitary. These observations suggest that, in both R. dybowskii and bullfrog, type 1 GnRHR is the receptor for hypothalamic GnRH ([Trp⁸]GnRH in R. dybowskii and mGnRH in the bullfrog) and is responsible for the control of pituitary gonadotrope cell activity, whereas type 2 and type 3 GnRHRs are the receptors for cGnRH-II that acts as a neurotransmitter and/or neuromodulator to regulate reproductive behavior. In support of this hypothesis, cGnRH-II has been found to modulate K⁺ currents in bullfrog sympathetic ganglia (26) and to stimulate reproductive behavior in female sparrow (27). However, we do not rule out a possible role of type 1 GnRHR in the brain and/or type 3 GnRHR in the pituitary because Northern and PCR analyses revealed expression, albeit at a low level, of dyGnRHR-1 in the brain and dyGnRHR-3 in the pituitary. As a matter of fact, we have found that mGnRH but not cGnRH-II can stimulate neurosteroid biosynthesis in the hypothalamic area in R. ridibunda (Beaujean, D., J. L. Do-Rego, J. Y. Seong, H. B. Kwou, and H. Vaudry, unpublished data), confirming the possible involvement of frog GnRHR-1 in neuromodulation. Reciprocally, we have observed a marked increase in bfGnRHR-3 gene expression in the pituitary during the prebreeding season (28), suggesting that bfGnRHR-3 could play a specific role in the control of FSH release. Indeed, although all GnRH variants can regulate the secretion of both LH and FSH, certain GnRH isoforms such as cGnRH-II and lamprey GnRH-III, preferentially stimulate FSH release (25, 29, 30). A second GnRHR has been recently characterized in the monkey (24, 25). This receptor is expressed throughout the brain and various peripheral tissues including the pituitary. Double immunohistochemical labeling of sheep pituitary with GnRHR-2 and LH antisera revealed that 69% of gonadotrope cells express GnRHR-2 (25). It is of interest to note that the monkey GnRHR-2 exhibits a higher degree of homology with dyGnRHR-3 than other frog GnRHRs. A recent phylogenetic tree analysis has confirmed that monkey GnRHR-2 is closest to frog GnRHR-3 (31). Considering the preferential sensitivity of frog GnRHR-3 to cGnRH-II (Ref. 14 and this study), it is conceivable that frog GnRHR-3 may play a role in the control of FSH release in the frog pituitary.

    The three dyGnRHRs showed a high degree of sequence similarity with their bfGnRHR counterparts and exhibited similar tissue distributions. When transfected in HeLa cells, each dyGnRHR showed similar ligand binding affinity and expression levels as its bfGnRHR ortholog. However, dyGnRHRs revealed different signaling ability and coupling efficiency when compared with their bfGnRHR counterparts. All frog GnRHRs except dyGnRHR-1 and dyGnRHR-2 activated both G_s and G_q/11-mediated signal transduction. Upon stimulation by GnRH, dyGnRHR-1 and dyGnRHR-2 significantly increased CRE-luc activity but failed to increase either GnRH-induced IP accumulation or c-fos-luc activity. Thus, dyGnRHR-1 and dyGnRHR-2 are the only GnRHRs that exclusively couple to G_s but not to G_q/11. It is still uncertain whether mammalian GnRHR couples to G_q/11 solely or to both G_s and G_q/11 (32, 33, 34). A recent study suggested that multiple signaling pathways downstream the mammalian GnRHR occur through exclusive coupling to G_q/11 (32). In this study, the authors did not observe a substantial increase in cAMP level or CRE-luc activity by GnRH challenges in certain cells. They suggested that an increase in cAMP levels in response to GnRH stimulation in other cells may originate from activation of adenylyl cylase-I that is triggered by increased intracellular Ca²⁺ level but not by the G_s. However, several other studies have shown that GnRH can cause a considerable increase in cAMP levels and CRE-luc activity (28, 33), and indicate that the first intracellular loop of the GnRHR may be responsible for G_s coupling (34). Consistent with the latter reports, we found that dyGnRHR-1 and dyGnRHR-2 can increase CRE-luc activity without activating G_q/11-mediated signaling. Interestingly, dyGnRHR-1 and dyGnRHR-2 had about 4- to 10-fold lower ligand sensitivity than their bfGnRHR counterparts in the CRE-luc reporter system. The decrease in ligand sensitivity may not be due to receptor expression or ligand affinity because the ligand binding assay revealed similar expression levels between dyGnRHRs and bfGnRHRs and no critical differences in dissociation constant values. Thus, the decreases in ligand sensitivity of dyGnRHR-1 and dyGnRHR-2 are suggestive of a lower coupling efficiency. In fact, mutation studies have previously shown that a decrease in coupling efficiency can occur without changes in expression levels and ligand affinity (22).

    We hypothesized that differences in ligand sensitivity and signal transduction coupling between dyGnRHR and bfGnRHR may result from amino acid substitutions in certain domains of frog GnRHRs. Chimeric receptors and amino acid exchanges of type 1 frog GnRHR clearly revealed some specific amino acids in ECL2 and TM VI are responsible for ligand sensitivities and signal transduction pathways. The increase in ligand sensitivity of bfBdy and A²⁰¹T dyGnRHR mutant suggests that Ala/Thr²⁰¹ in ECL2 of the type 1 receptor is critical for ligand sensitivity. It is of interest to note that Thr²⁰¹ in ECL2 of the GnRHR is highly conserved throughout the mammalian and nonmammalian species. Thus, it is plausible that the change of Thr²⁰¹ to Ala in dyGnRHR-1 critically modifies ligand-receptor interaction or receptor conformation after ligand binding. Leu/Phe²⁹⁰ in TM VI is also important for ligand sensitivity because the dyBbf and L²⁹⁰F mutant receptors exhibited increases in ligand sensitivity. Moreover, L²⁹⁰F mutant significantly increased IP production and c-fos-luc activity in response to GnRH. This finding was further confirmed using bfGnRHR-1 mutants. F²⁹⁰LY mutant remarkably decreased IP production and c-fos-luc activity. A complete loss of ability to induce IP production and c-fos-luc activity was observed in a double mutant, F²⁹⁰LY²⁹⁹F. TM VI of the mammalian and nonmammalian GnRH receptors is highly conserved and encompasses a high proportion of aromatic amino acids. Although the role of TM VI of GnRHR is not as well understood as that of the other TMs, recent studies indicate that TM VI may be important for receptor expression, ligand recognition, and signaling efficiency (35, 36, 37). In particular, a point mutation in TM VI (Cys²⁷⁹ "->" Tyr) has been shown to cause GnRH refractoriness in a patient with idiopathic hypogonadotropic hypogonadism (35). Similarly, a single amino acid substitution of Phe²⁷² to Leu or Tyr in the human GnRHR results in overexpression or lower expression, respectively (36). The CWTPYYLLGL/IWYWF motif in TM VI (position 279–292 in the human GnRHR, 278–291 in the rat GnRHR, and 285–298 in dyGnRHR-1) is remarkably well conserved. This motif may be kinked near the conserved Pro²⁸⁸ such that an aromatic cage may form in this motif. The Trp²⁷⁹ residue in the rat GnRHR appears an important site for ligand affinity and signal transduction because a single amino acid substitution of Trp²⁷⁹ to Ser or Arg results in a marked reduction in ligand binding and GnRH-induced IP production (37). Computer modeling revealed that Trp²⁷⁹ moiety may directly interact with the Trp³ residue of GnRH (37). Thus, the observation that L²⁹⁰F dyGnRHR-1 mutant has an increased ability to stimulate IP production is quite interesting. Within the CWTPYYLLGL/IWYWF motif, the Tyr residue (underlined) was changed to Phe and Leu in bfGnRHR-1 and dyGnRHR-1, respectively. Phe is structurally closer to Tyr than Leu as both Phe and Tyr, but not Leu possess an aromatic ring. Because both bfGnRHR-1 and dyGnRHR-1 with L²⁹⁰F mutation exhibited higher ability to induce IP production and c-fos-luc activity, an aromatic residue at this position appears to be important for G_q/11-mediated signal transduction. In mammalian GnRHRs, a Tyr²⁸⁴ residue at the same position of Leu/Phe²⁹⁰ of frog type 1 receptor is also important for signal transduction. In the human GnRHR, a change of the Tyr²⁸⁴ residue to Ala lost either ligand binding or signal transduction (38). Moreover, a change of this Tyr residue to Leu in the rat GnRHR decreased c-fos promoter-driven gene activation by 50% (Oh, D. Y., H. B. Kwon, and J. Y. Seong, unpublished data). It is well known that aromatic interaction within the TM structure is important for ligand binding and/or signaling efficacy of GnRHR (33) and other G protein-coupled receptors (39, 40). Thus, it is possible that aromatic interaction of Tyr or Phe with other aromatic amino acids either adjacent to this amino acid or in other TMs is critical for IP production. Because no significant differences in receptor expression were observed between bfGnRHR-1 and dyGnRHR-1, it is possible that the Leu to Phe change may play a role in stabilizing the active state of ligand-receptor-G protein complex. However, this possibility should be further clarified.

    In summary, the data presented here clearly demonstrate that three different types of the GnRHR are present in Rana species. It is also of particular interest that each type of the GnRHR in the same genus exhibits species specificity in ligand recognition and signal transduction coupling. Understanding the structure-function relationships of the various GnRHRs should open new avenues for development of selective agonists with therapeutic value.3, http://www.100md.com

    Received July 5, 2002.3, http://www.100md.com

    Accepted for publication October 11, 2002.3, http://www.100md.com

    References3, http://www.100md.com

    Sealfon SC, Weinstein H, Millar RP 1997 Molecular mechanisms of ligand interaction with the gonadotropin-releasing hormone receptor. Endocr Rev 18:180–2053, http://www.100md.com

    Schally AV, Arimura A, Kastin AJ, Matsuo H, Baba Y, Redding TW, Nair RM, Debeljuk L, White WF 1971 Gonadotropin-releasing hormone: one polypeptide regulates secretion of luteinizing and follicle-stimulating hormones. Science 173:1036–1038

    Burgus R, Butcher M, Amoss M, Ling N, Monahan M, Rivier J, Fellows R, Blackwell R, Vale W, Guillemin R 1972 Primary structure of the ovine hypothalamic luteinizing hormone-releasing factor (LRF) (LH-hypothalamus-LRF-gas chromatography-mass spectrometry-decapeptide-Edman degradation). Proc Natl Acad Sci USA 69:278–282[6q{'e, http://www.100md.com

    Fernald RD, White RB 1999 Gonadotropin-releasing hormone genes: phylogeny, structure, and functions. Front Neuroendocrinol 20:224–240[6q{'e, http://www.100md.com

    Miyamoto K, Hasegawa Y, Nomura M, Igarashi M, Kangawa K, Matsuo H 1984 Identification of the second gonadotropin-releasing hormone in chicken hypothalamus: evidence that gonadotropin secretion is probably controlled by two distinct gonadotropin-releasing hormones in avian species. Proc Natl Acad Sci USA 81:3874–3878[6q{'e, http://www.100md.com

    White RB, Eisen JA, Kasten TL, Fernald RD 1998 Second gene for gonadotropin-releasing hormone in humans. Proc Natl Acad Sci USA 95:305–309[6q{'e, http://www.100md.com

    Conlon JM, Collin F, Chiang YC, Sower SA, Vaudry H 1993 Two molecular forms of gonadotropin-releasing hormone from the brain of the frog, Rana ridibunda: purification, characterization, and distribution. Endocrinology 132:2117–2123

    Wang L, Yoo MS, Kang HM, Im WB, Choi HS, Bogerd J, Kwon HB 2001 Cloning and characterization of cDNAs encoding the GnRH1 and GnRH2 precursors from bullfrog (Rana catesbeiana). J Exp Zool 289:190–201p[yva}l, http://www.100md.com

    Yoo MS, Kang HM, Choi HS, Kim JW, Troskie BE, Millar RP, Kwon HB 2000 Molecular cloning, distribution and pharmacological characterization of a novel gonadotropin-releasing hormone ([Trp⁸] GnRH) in frog brain. Mol Cell Endocrinol 164:197–204p[yva}l, http://www.100md.com

    Suzuki M, Hyodo S, Kobayashi M, Aida K, Urano A 1992 Characterization and localization of mRNA encoding the salmon-type gonadotrophin-releasing hormone precursor of the masu salmon. J Mol Endocrinol 9:73–82p[yva}l, http://www.100md.com

    Powell JF, Zohar Y, Elizur A, Park M, Fischer WH, Craig AG, Rivier JE, Lovejoy DA, Sherwood NM 1994 Three forms of gonadotropin-releasing hormone characterized from brains of one species. Proc Natl Acad Sci USA 91:12081–12085p[yva}l, http://www.100md.com

    Yahalom D, Chen A, Ben-Aroya N, Rahimipour S, Kaganovsky E, Okon E, Fridkin M, Koch Y 1999 The gonadotropin-releasing hormone family of neuropeptides in the brain of human, bovine and rat: identification of a third isoform. FEBS Lett 463:289–294

    Troskie B, Illing N, Rumbak E, Sun YM, Hapgood J, Sealfon S, Conklin D, Millar R 1998 Identification of three putative GnRH receptor subtypes in vertebrates. Gen Comp Endocrinol 112:296–302r7, http://www.100md.com

    Wang L, Bogerd J, Choi HS, Seong JY, Soh JM, Chun SY, Blomenröhr M, Troskie BE, Millar RP, Yu WH, McCann SM, Kwon HB 2001 Three distinct types of GnRH receptor characterized in the bullfrog. Proc Natl Acad Sci USA 98:361–366r7, http://www.100md.com

    Reinhart J, Mertz LM, Catt KJ 1992 Molecular cloning and expression of cDNA encoding the murine gonadotropin-releasing hormone receptor. J Biol Chem 267:21281–21284r7, http://www.100md.com

    Tsutsumi M, Zhou W, Millar RP, Mellon PL, Roberts JL, Flanagan CA, Dong K, Gillo B, Sealfon SC 1992 Cloning and functional expression of a mouse gonadotropin-releasing hormone receptor. Mol Endocrinol 6:1163–1169r7, http://www.100md.com

    Tensen C, Okuzawa K, Blomenröhr M, Rebers F, Leurs R, Bogerd J, Schulz R, Goos H 1997 Distinct efficacies for two endogenous ligands on a single cognate gonadoliberin receptor. Eur J Biochem 243:134–140

    Illing N, Troskie BE, Nahorniak CS, Hapgood JP, Peter RE, Millar RP 1999 Two gonadotropin-releasing hormone receptor subtypes with distinct ligand selectivity and differential distribution in brain and pituitary in the goldfish (Carassius auratus). Proc Natl Acad Sci USA 96:2526–2531oy|l*j+, http://www.100md.com

    Troskie BE, Hapgood JP, Millar RP, Illing N 2000 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. Endocrinology 141:1764–1771oy|l*j+, http://www.100md.com

    Heding A, Vrecl M, Bogerd J, McGregor A, Sellar R, Taylor PL, Eidne KA 1998 Gonadotropin-releasing hormone receptors with intracellular carboxyl-terminal tails undergo acute desensitization of total inositol phosphate production and exhibit accelerated internalization kinetics. J Biol Chem 273:11472–11477oy|l*j+, http://www.100md.com

    Lin X, Janovick JA, Brothers S, Blomenröhr M, Bogerd J, Conn PM 1998 Addition of catfish gonadotropin-releasing hormone (GnRH) receptor intracellular carboxyl-terminal tail to rat GnRH receptor alters receptor expression and regulation. Mol Endocrinol 12:161–171

    Flanagan CA, Zhou W, Chi L, Yuen T, Rodic V, Robertson D, Johnson M, Holland P, Millar RP, Weinstein H, Mitchell R, Sealfon SC 1999 The functional microdomain in transmembrane helices 2 and 7 regulates expression, activation, and coupling pathways of the gonadotropin-releasing hormone receptor. J Biol Chem 274:28880–288864, 百拇医药

    Conn PM, Crowley Jr WF 1994 Gonadotropin-releasing hormone and its analogs. Annu Rev Med 45:391–4054, 百拇医药

    Neill JD, Duck LW, Sellers JC, Musgrove LC 2001 A gonadotropin-releasing hormone (GnRH) receptor specific for GnRH II in primates. Biochem Biophys Res Commun 282:1012–10184, 百拇医药

    Millar R, Lowe S, Conklin D, Pawson A, Maudsley S, Troskie B, Ott T, Millar M, Lincoln G, Sellar R, Faurholm B, Scobie G, Kuestner R, Terasawa E, Katz A 2001 A novel mammalian receptor for the evolutionarily conserved type II GnRH. Proc Natl Acad Sci USA 98:9636–96414, 百拇医药

    Troskie B, King JA, Millar RP, Peng YY, Kim J, Figueras H, Illing N 1997 Chicken GnRH II-like peptides and a GnRH receptor selective for chicken GnRH II in amphibian sympathetic ganglia. Neuroendocrinology 65:396–402

    Maney DL, Richardson RD, Wingfield JC 1997 Central administration of chicken gonadotropin-releasing hormone-II enhances courtship behaviour in a female sparrow. Horm Behav 32:11–18&n, 百拇医药

    Wang L, Oh DY, Bogerd J, Choi HS, Ahn RS, Seong JY, Kwon HB 2001 Inhibitory activity of alternative splice variants of the bullfrog GnRH receptor-3 on wild-type receptor signaling. Endocrinology 142:4015–4025&n, 百拇医药

    Millar RP, Milton RC, Follett BK, King JA 1986 Receptor binding and gonadotropin-releasing activity of a novel chicken gonadotropin-releasing hormone ([His⁵, Trp⁷, Tyr⁸]GnRH) and a D-Arg⁶ analog. Endocrinology 119: 224–231&n, 百拇医药

    Yu WH, Karanth S, Walczewska A, Sower SA, McCann SM 1997 A hypothalamic follicle-stimulating hormone-releasing decapeptide in the rat. Proc Natl Acad Sci USA 94:9499–9503&n, 百拇医药

    Okubo K, Nagata S, Ko R, Kataoka H, Yoshiura Y, Mitani H, Kondo M, Naruse K, Shima A, Aida K 2001 Identification and characterization of two distinct GnRH receptor subtypes in a teleost, the medaka Oryzias latipes. Endocrinology 142:4729–4739

    Grosse R, Schmid A, Schoneberg T, Herrlich A, Muhn P, Schultz G, Gudermann T 2000 Gonadotropin-releasing hormone receptor initiates multiple signaling pathways by exclusively coupling to G_q/11 proteins. J Biol Chem 275:9193–9200:ac@#, 百拇医药

    Hoffmann SH, ter Laak T, Kuhne R, Reilander H, Beckers T 2000 Residues within transmembrane helices 2 and 5 of the human gonadotropin-releasing hormone receptor contribute to agonist and antagonist binding. Mol Endocrinol 14:1099–1115:ac@#, 百拇医药

    Arora KK, Krsmanovic LZ, Mores N, O’Farrell H, Catt KJ 1998 Mediation of cyclic AMP signaling by the first intracellular loop of the gonadotropin-releasing hormone receptor. J Biol Chem 273:25581–25586:ac@#, 百拇医药

    Beranova M, Oliveira LM, Bedecarrats GY, Schipani E, Vallejo M, Ammini AC, Quintos JB, Hall JE, Martin KA, Hayes FJ, Pitteloud N, Kaiser UB, Crowley Jr WF, Seminara SB 2001 Prevalence, phenotypic spectrum, and modes of inheritance of gonadotropin-releasing hormone receptor mutations in idiopathic hypogonadotropic hypogonadism. J Clin Endocrinol Metab 86:1580–1588

    Myburgh DB, Pawson AJ, Davidson JS, Flanagan CA, Millar RP, Hapgood JP 1998 A single amino acid substitution in transmembrane helix VI results in overexpression of the human GnRH receptor. Eur J Endocrinol 139:438–447t+ow?3:, 百拇医药

    Chauvin S, Berault A, Lerrant Y, Hibert M, Counis R 2000 Functional importance of transmembrane helix 6 Trp²⁷⁹ and exoloop 3 Val²⁹⁹ of rat gonadotropin-releasing hormone receptor. Mol Pharmacol 57:625–633t+ow?3:, 百拇医药

    Hövelmann S, Hoffmann SH, Kuhne R, ter Laak T, Reilander H, Beckers T 2002 Impact of aromatic residues within transmembrane helix 6 of the human gonadotropin-releasing hormone receptor upon agonist and antagonist binding. Biochemistry 41:1129–1136t+ow?3:, 百拇医药

    Lu ZL, Saldanha JW, Hulme EC 2001 Transmembrane domains 4 and 7 of the M₁ muscarinic acetylcholine receptor are critical for ligand binding and the receptor activation switch. J Biol Chem 276:34098–34104t+ow?3:, 百拇医药

    Labrou NE, Bhogal N, Hurrell CR, Findlay JB 2001 Interaction of met297 in the seventh transmembrane segment of the tachykinin nk2 receptor with neurokinin a. J Biol Chem 276:37944–37949(Jae Young Seong Li Wang Da Young Oh Oim Yun Kaushik Maiti Jian Hua Li Jae Mok Soh Hueng Sik Choi Kyu)

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