The human gonadotropin-releasing hormone (GnRH) receptor gene: cloning, genomic organization and chromosomal assignment

ELSEVIER

Molecular and Cellular Endocrinology 103 (1994) R l -R6

~ Molecularand Cellular Endocrinology

Rapid paper

The human gonadotropin-releasing hormone (GnRH) receptor gene: cloning, genomic organization and chromosomal assignment N a n c y C. F a n a, E u i - B a e J e u n g a, C h u n P e n g a, J a n I. O l o f s s o n b, J o h n K r i s i n g e r a, P e t e r C . K . L e u n g a,* aDepartment of Obstetrics and Gynaecology, Universityof British Columbia, Vancouver, B.C. V6H 3V5 Canada bDepartment of Physiology, Universityof Umed, S-901 87 Umed, Sweden

Received 21 April 1994; accepted 11 May 1994

Abstract The cDNA encoding the gonadotropin-releasing hormone (GnRH) receptor has recently been cloned and characterized in several species, including human. To determine the structure of the gene encoding the human GnRH receptor, we have screened a human genomic library and isolated seven positive clones, using cDNA probes derived from a human pituitary cDNA library. The isolated genomic clone contains the entire protein coding region of the GnRH receptor which is distributed between three exons and spans over 18.9 kb. Sequence analysis and restriction endonuclease mapping revealed the presence of two introns of 4.2 and 5.0 kb, respectively, both located within the open reading frame, designating the human GnRH receptor gene to the intron-containing class of the G-protein coupled receptor superfamily. Genomic Southern blot analysis indicated the presence of a single copy of the gene encoding for the GnRH receptor within the human genome. Using DNA from human-hamster somatic hybrid cell lines, the GnRH receptor gene was assigned to human chromosome 4, by means of PCR. The present study represents the first report on the GnRH receptor gene and its partial characterization should facilitate further investigation of the mechanisms by which expression of this gene is regulated. Keywords: Gonadotropin-releasing hormone receptor; Gene structure (human); Chromosome mapping

1. Introduction Gonadotropin-releasing hormone (GnRH) plays a fundamental role in the control of reproduction. This decapeptide stimulates the synthesis and release of both luteinizing hormone and follicle stimulating hormone from the anterior pituitary where specific membrane bound receptors for GnRH are also found (for reviews, see Clayton et al., 1989; Braden et al., 1992; Sherwood et al., 1993). In addition, GnRH acts locally to regulate human chorionic gonadotropin secretion in the placenta (Petraglia et al., 1990; Currie et al., 1992) and steroidogenesis in the ovary (Hsueh and Jones, 1981; Leung and Steele, 1992).

* Corresponding author, Department of Obstetrics and Gynaecology, University of British Columbia, Room 2H30, 4490 Oak Street, Vancouver, B.C. Canada V6H 3V5. Tel. (604)875-2718. Fax (604)875-2717.

Recently, cDNAs encoding the GnRH receptor have been cloned and characterized in pituitary cells from several species, including mouse (Tsutsumi et al., 1992; Reinhart et al., 1992), rat (Kaiser et al., 1992; Eidne et al., 1992; Perrin et al., 1993), sheep (Brooks et al., 1993; Illing et al., 1993), cow (Kakar et al., 1993) and human (Kakar et al., 1992; Chi et al., 1993). Highly conserved between these species (>85%), the human transcript encodes a 328 amino acid protein. The common feature of these sequences revealed seven putative transmembrane domains characteristic of G-protein coupled receptors and the absence of a cytosolic COOH-terminal tail. Besides the classical localization of GnRH receptor gene expression to anterior pituitary gonadotrophs, mRNAs encoding the GnRH receptor have also been demonstrated in a number of gonadal tissues, including ovary and testis, but not rat placenta (Kakar et al., 1992; Kaiser et al., 1992; Olofsson et al., 1994). However, the structure for the gene has hitherto remained unidentified.

0167-8140/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0167-8140(94)03330-K

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To elucidate the structural organization and establish a foundation for further studies on transcriptional regulation of the GnRH receptor gene, we have successfully isolated the gene encoding the human GnRH receptor and describe herein its partial structural organization and chromosomal assignment. 2. Materials and methods 2.1. Isolation of the human GnRH receptor genomic clones

A human genomic library constructed in FIXII (obtained from Stratagene, La Jolla, CA) was screened using a human pituitary GnRH receptor cDNA as a probe. Approximately 1.2 × 106 recombinant A-plaques were plated and transferred to Hybond-N nylon membranes (Amersham, Oakville, ON, Canada). A 760 bp cDNAfragment corresponding to nucleotide +201 to +960 was obtained from screening of a human pituitary cDNA library (Clontech, Palo Alto, CA) using a 416 bp rat cDNA PstI-fragment as a probe (Olofsson et al., 1994; Peng et al., unpublished results). Probes were labelled with [a32p]dCTP (Amersham) by the random priming method and hybridized with filters under high stringency conditions. After three rounds of screening with the human cDNA probe, phage DNA was isolated from the positive clones, digested with restriction enzymes and the fragments were electrophoresed on a 0.8% agarose gel and analyzed by Southern blot hybridization using the cDNA as well as synthetic oligonucleotide probes representing the 5' and 3' end of the published cDNA sequences (Chi et al., 1993; Kakar et al., 1993), using similar protocols as described previously (Jeung et al., 1994). Three EcoRI fragments were then subcloned into pUC19 vectors and sequenced. 2.2. Sequence analysis and alignment

DNA fragments were sequenced by the dideoxy chain termination method (Sanger et al., 1977) using a T7 DNA polymerase sequencing kit (Pharmacia, Ltd, Uppsala, Sweden) and internal synthetic oligonucleotide primers as well as universal forward and reverse primers. The nucleotide sequence of the GnRH receptor gene was then compared with the GnRH receptor cDNA to determine the location of exon-intron boundaries using software from the Genetics Computer Group Inc. (Madison, WI). 2.3. Genomic Southern Blot analysis

Genomic DNA was isolated from human placenta using standard methods (Sambrook et al., 1989). Ten micrograms of human genomic DNA was digested with restriction endonucleases (EcoRI, HindlII, BamHI and PstI), electrophoresed on a 0.8% agarose gel, and transferred to a nylon membrane. The filter was then hybridized with a [a-32p]dCTP-labelled 760bp human GnRH receptor cDNA (probe A, Fig. 1) and PstI-digested fragments,

396 bp (probe B) and 364 bp in length (probe C), respectively. 2.4. Chromosomal assignment

The chromosomal localization of the human GnRH receptor gene was determined by polymerase chain reaction (PCR) technique using DNA from 25 human-hamster somatic hybrid cell lines (BIOS laboratories, New Haven, CT). The primers (sense; 5'-AAGTAGGATTTACACTTAAGT and antisense; 5"-AACTATAGGAGGGAAAGTTGA, with nucleotide positions +1090-1110 and +1466-1486, respectively) were used. The reaction mixtures, containing 200 ng of hybrid cell line genomic DNA, were subjected to 30 cycles of PCR using similar conditions as previously described (Jeung et al., 1994). The reaction products were then electrophoresed on a 1.0% agarose gel, visualized by ethidium bromide staining and blotted to a nylon membrane. The filter was then hybridized with a [y-32p]ATP end-labelled internal oligonucleotide (5'-TAAACTCAGCATGCTTF, +13611377) probe. 3. Results and discussion 3.1. Isolation and characterization of the human GnRH receptor gene

Upon screening of the human genomic library with the 760 bp human cDNA probe under high stringency hybridization conditions, 12 genomic clones were isolated from an initial screening of approximately 1.2 x 106 plaques. After three consecutive rounds of screening, positive clones were purified to homogeneity and ADNA was prepared and digested with different restriction enzymes. DNA fragments were analyzed by Southern blot using the human GnRH receptor cDNA and also by oligonucleotides corresponding to the 5' and 3' untranslated regions of GnRH receptor cDNA sequence to determine the position of the A-clones with respect to the 5' and 3' end of the gene. One A-clone contained a 7.7 kb EcoRIfragment corresponding to the 5'-end of the gene and another A-clone contained both a 7.4 kb and 3.8 kb EcoRIfragment, respectively, all of which hybridized with either the cDNA or oligonucleotide probe(s). These fragments were subsequently subcloned into pUC19 vectors and sequenced. As shown in Fig. 1, the gene consists of at least three exons and two introns and spans over 18.9 kb. Together, the ).-clones containing the entire coding region has been sequenced and found to be identical to the published human GnRH receptor cDNA sequences (Chi et al., 1993; Kakar et al., 1993) and therefore, will not be repeated here. Exon I carries the 5'-untranslated region and part of the open reading frame encompassing transmembrane domains I-III and a portion of transmembrane domain IV. Since the transcriptional initiation site(s) is at present unknown, the exact size of exon I cannot be determined. In

N.C. Fan et al. / Mol. Cell. Endocrinol. 103 (1994) R1-R6

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clone 1

clone2

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Fig. 1. Schematic representation of the human GnRH receptor gene. (A) Organization of three genomic subclones. E; EcoRl. (B) The strategy for sequencing. Boxes represent exons and arrows indicate the extent of sequence obtained. (C) Exon-intron localization. (D) The structure of human GnRH receptor cDNA. Open box indicates the protein coding regions and hatched boxes are the putative transmembrane domains. (E) The relative positions of the human cDNA-probes used for screening and genomic Southern blot analysis. addition, the possibility of having additional 5'-exons cannot be ruled out. The second exon is only 219 bp in size and codes for the remainder of the IVth together with the Vth transmembrane domain of the deduced amino acid sequence. The third exon encodes the COOH-terminal part of the open reading frame and the 3' untranslated region. The 3' sequence obtained so far (+2233) contains no classical polyadenylation signal ( A A T A A A ) and extends further compared to the longest published (Chi et al., 1993) 3' untranslated region of the human GnRH receptor cDNA (=1.1 kb). Based on the size of the predominant transcript of 4.7 kb (Chi et al., 1993; own unpublished results), an additional 3' untranslated sequence can be predicted. Therefore, the exact size of exon III is at present not known. Of particular interest is the finding of two introns present within the protein coding region (Table 1), since many G-protein coupled genes examined to date are intronless (Probst et al., 1992). The first intron was determined to localize within the IVth transmembrane domain

and the second intron was found in the third intracellular loop of the deduced amino acid sequence. Amongst the genes encoding G-protein coupled receptors, introns (if present) tend to be positioned between the transmembrane domains, but there are several known exceptions to this rule as exemplified by the human rhodopsin and opsin genes (Nathans et al., 1984, 1986) and the human 5-HT 2 receptor gene (Chen et al., 1992).

3.2. Genomic southern blot analysis Genomic Southern blot analysis was performed to detect the GnRH receptor gene directly in human genomic DNA. Genomic DNA from human placenta was digested with one of four restriction endonucleases; EcoRI, BamHI, HindlII and PstI. Each digest generated multiple fragments that hybridized to the 760 bp cDNA probe (A). However, when this probe was digested with PstI to generate a 5'-oriented 396 bp probe (B) and a 3'-oriented 364 bp probe (C), the number of DNA fragments hybridizing to each probe decreased (Fig. 2). For example, three fragments (7.7, 7.4 and 3.8 kb, respectively) hybridized

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Table 1 Exon-intron organization of the human GnRH receptor gene Exon

Exon size (bp)

Sequence of exon-intron junction 5'-Boundary

Intron (kb)

3'-Boundary

1

>600

A CCA CAG (+522) Gin (174)

gtgaa . . . . 4.2 ...tacag

TTA TAC A Leu (175)

2

219

CCC CAC G (+682) Glu (248)

gtatg... =5.0 ...aacag

AA CTA CAA Leu (249)

3

> 1540

AAA ATG G...(+2223)

The splice-junctions share the consensus sequences for donor and acceptor sites (Breathnach and Chambon, 1981). Numbers in parentheses are relative to the translation initiation codon.

to probe A (Fig. 2A), but only the 7.7 kb fragment hybridized to probe B (Fig. 2B) whereas the 7.4 and 3.8 kb moieties hybridized to probe C (Fig. 2C). The hybridization pattern of the genomic Southern blot is in agreement with the restriction map obtained from the isolated clones. Furthermore, the results clearly indicate the presence of a single copy of the GnRH receptor gene in the human genome. Hybridization at lower stringency failed to detect additional related sequences (data not shown).

3.3. Chromosome assignment DNA isolated from human-hamster somatic hybrid cell A

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lines and their parental cell lines were used to examine the presence of the GnRH receptor gene by PCR using primers specific for the human GnRH receptor. As shown in Fig. 3, the PCR detected a major product of expected size (397 bp) from a human cell line and two hybrid cell lines, whereas the similar product was not observed in a hamster cell line and the rest of the hybrid cell lines. When the PCR product was transferred onto a nylon membrane and hybridized with an internal oligonucleotide probe, only the human cell line and two hybrid cell lines (nos. 852 and 1079) showed positive signals, thus confirming that the product formed is the authentic GnRH receptor (data not shown). Comparison with the panel of B

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Fig. 2. Genomic Southern blot analysis of the human GnRH receptor gene. Ten micrograms of human genomic DNA was digested with each EcoRI, BamHI, HindllI and PstI and probed with either cDNA probe A, B or C (for positions see Fig. 1).

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Fig. 3. Chromosomal assignment of the human GnRH receptor gene to chromosome4. DNA from 25 human-hamster somatic hybrids cell lines and their parental cell lines were amplified by 30 cycles of PCR using primers derived from the human GnRH receptor cDNA sequence (see Section 2). A 397 bp product was obtained from two hybrid cell lines (nos. 852 and 1079) and the human parental cell line but not from the hamster parental cell line and a representative hybrid cell line (no. 983). human chromosomal components provided for each cell line identified chromosome number 4 as the carrier of the human GnRH receptor gene. The assignment of the human GnRH receptor gene to chromosome 4 is identical with the localization of several other genes belonging to the superfamily of G-protein coupled receptors such as a2-C4 adrenergic receptor (Kobilka et al., 1987), cholecystokinin receptor A (de Weerth et al., 1993), D5 dopamine receptor (Polymeropoulos et al., 1991; Eubanks et al., 1992), endothelin-A receptor (Hosoda et al., 1992) and neuropeptide Y Y1 receptor (Herzog et al., 1993). Further analyses on the subchromosomal localization of the human GnRH receptor gene may enable us to identify closely linked genes. In conclusion, the partial structure of the human GnRH receptor gene was determined and the isolated genomic clones cover the entire protein coding region. Furthermore, the presence of two introns splicing the open reading frame, designates this gene to the intron-containing class of G-protein coupled receptor genes. In addition, genomic Southern blot analysis revealed that the GnRH receptor gene is unique in the human genome and has been determined to localize on chromosome 4. To our knowledge, this is the first report on the isolation, genomic organization and chromosomal assignment for the GnRH receptor gene and will greatly facilitate future

investigation concerning the molecular regulation of the GnRH receptor as well as the evolution of the G-protein coupled gene superfamily.

Acknowledgements This work was supported by the Medical Research Council of Canada (No. MT7-711). C. Peng is supported by a postdoctoral fellowship from the National Sciences and Engineering Research Council of Canada. J.I. Olofsson is the recipient of a scholarship from the Swedish Medical Research Council and a travel grant from the Swedish Society of Medicine. P.C.K. Leung is a Career Investigator of the British Columbia Children's Hospital.

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