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Cloning and Characterization of the 5′-Flanking Region of the Human Growth Hormone Secretagogue Receptor Gene

1998; Elsevier BV; Volume: 273; Issue: 51 Linguagem: Inglês

10.1074/jbc.273.51.33885

ISSN

1083-351X

Autores

Hidesuke Kaji, Shigeru Tai, Yasuhiko Okimura, Genzo Iguchi, Yutaka Takahashi, Hiromi Abe, Kazuo Chihara,

Tópico(s)

Regulation of Appetite and Obesity

Resumo

Recently, the growth hormone secretagogue receptor (GHS-R) cDNA has been isolated from the pituitary and hypothalamus. To evaluate the regulation of human (h) GHS-R gene expression, we cloned the hGHS-R gene containing the 5′-flanking region of 0.6–2.9 kilobase pairs. Analysis of the hGHS-R transcripts with 5′-rapid amplification of cDNA ends suggested that the putative transcription initiation site was approximately −453 base pairs upstream of the translation initiation site (+1). There is no typical TATA, CAAT, or GC box but an initiator-like sequence and putative binding sites for several transcription factors around the putative transcription start site. The 5′-flanking region inserted into a luciferase reporter vector had promoter activity in GH3 cells but had activity indistinguishable from background in HeLa or EP1 cells. The hGHS-R promoter activity in GH3 cells increased by deletion of nucleotides from −1224 to −734, whereas it was decreased by further deletion from −734 to −608. Knowledge of the promoter region of the hGHS-R gene will facilitate elucidation of its transcriptional control. Recently, the growth hormone secretagogue receptor (GHS-R) cDNA has been isolated from the pituitary and hypothalamus. To evaluate the regulation of human (h) GHS-R gene expression, we cloned the hGHS-R gene containing the 5′-flanking region of 0.6–2.9 kilobase pairs. Analysis of the hGHS-R transcripts with 5′-rapid amplification of cDNA ends suggested that the putative transcription initiation site was approximately −453 base pairs upstream of the translation initiation site (+1). There is no typical TATA, CAAT, or GC box but an initiator-like sequence and putative binding sites for several transcription factors around the putative transcription start site. The 5′-flanking region inserted into a luciferase reporter vector had promoter activity in GH3 cells but had activity indistinguishable from background in HeLa or EP1 cells. The hGHS-R promoter activity in GH3 cells increased by deletion of nucleotides from −1224 to −734, whereas it was decreased by further deletion from −734 to −608. Knowledge of the promoter region of the hGHS-R gene will facilitate elucidation of its transcriptional control. growth hormone GHS receptor human GHS-R fetal calf serum prolactin polymerase chain reaction rapid amplification of cDNA ends base pair(s) kilobase pair(s) initiator basic helix-loop-helix pituitary homeobox 1 luteinizing hormone signal transducers and activators of transcription. Growth hormone (GH)1 secretion is regulated mainly by the hypothalamic stimulatory factor, GH-releasing hormone, and the inhibitory factor, somatostatin. On the other hand, GH secretagogues have been developed as a small synthetic peptide, GH-releasing peptide (1Momany F.A. Bowers C.Y. Reynolds G.A. Hong A. Newlander K. Endocrinology. 1984; 114: 1531-1536Crossref PubMed Scopus (175) Google Scholar), and non-peptides, L-692,429 (2Smith R.G. Chen K. Schoen W.R. Pong S-S. Hickey G.J. Jacks T.M. Butler B.S. Chan W.W-S. Chaung L-Y.P. Judith F. Taylor A.M. Wyvratt Jr., M.J. Fisher M.H. Science. 1993; 260: 1640-1643Crossref PubMed Scopus (327) Google Scholar) and MK-0677 (3Patchett A.A. Nargund R.P. Tata J.R. Chen M-H. Barakat K.J. Johnston D.B.R. Chen K. Chan W.W-S. Butler B.S. Hickey G.J. Jacks T.M. Scleim K. Pong S-S. Chaung L-Y.P. Chen H.Y. Fraizier E. Leung K.H. Chui S.-H.L. Smith R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7001-7005Crossref PubMed Scopus (337) Google Scholar), with potent GH-secreting activity, especially in vivo and in humans. The recent cloning of the human, porcine (4Howard A.D. Feighner S.D. Cully D.F. Arena J.P. Liberator P.A. Rosenblum C.I. Hamelin M. Hreniuk D.L. Palyha O.C. Anderson J. Paress P.S. Diaz C. Chou M. Liu K.K. McKee K.K. Pong S-S. Chaung L-Y.P. Elbrecht A. Dashkevicz M. Heavens R. Rigby M. Sirinathsinghji D.J.S. Dean D.C. Mellilo D.G. Patchett A.A. Nargund R.P. Griffin P.R. Demartino J.A. Guputa S.K. Schaeffer J.M. Smith R.G. Van der Ploeg L.H.T. Science. 1996; 273: 974-977Crossref PubMed Scopus (1787) Google Scholar), and rat (5McKee K.K. Palyha O.C. Feighner S.D. Hreniuk D.L. Tan C.P. Phillips M.S. Smith R.G. Van der Ploeg L.H.T. Howard A.D. Mol. Endocrinol. 1997; 11: 415-423Crossref PubMed Scopus (289) Google Scholar) GHS receptor (GHS-R) cDNA has suggested an additional physiological regulation for GH release. GHS-R mRNA is expressed not only in the pituitary and hypothalamus but also in the hippocampus, pancreas (6Guan X-M., Yu,H. Palyha O.C. Mckee K.K. Feighner S.D. Sirinathsinghji D.J.S. Smith R.G. Van der Ploeg L.H.T. Howard A.D. Mol. Brain Res. 1997; 48: 23-29Crossref PubMed Scopus (971) Google Scholar), and neuroendocrine tumors (7de Keyzer Y. Lenne F. Bertagna X. Eur. J. Endocrinol. 1997; 137: 715-718Crossref PubMed Scopus (50) Google Scholar), including human somatotropinomas and rat GH3 cells (8Adams E.F. Huang B. Buchfelder M. Howard A. Smith R.G. Feighner S.D. Van der Pleog L.H.T. Bowers C.Y. Fahlbush R. J. Clin. Endocrinol. Metab. 1998; 83: 638-642PubMed Google Scholar). There are still only a few reports about the regulation of GHS-R expression. Bennettet al. (9Bennett P.A. Thomas G.B. Howard A.D. Feighner S.D. Van der Ploeg L.H.T. Smith R.G. Robinson I.C.A.F. Endocrinology. 1997; 138: 4552-4557Crossref PubMed Scopus (168) Google Scholar) have recently reported that GHS-R expression in the hypothalamus was markedly increased in dw/dw dwarf rats and was down-regulated in dw/dw rats treated with GH. They have also reported that GHS-R mRNA expression in the ventromedial nucleus of the hypothalamus was lower in male than in female rats.To understand the transcriptional regulation of the human GHS-R (hGHS-R) gene expression, we have cloned and characterized the 5′-flanking region of the hGHS-R gene.DISCUSSIONThe present study is the first report of a cloning and characterization of a human genomic DNA fragment containing the 5′-flanking region of the hGHS-R gene. When the sequence of the 5′-flanking region of the hGHS-R gene was compared with the upstream sequence of the hGHS-R cDNA, a single exon upstream of the coding exon was identified as shown for other human G protein-coupled receptor genes such as those for the M2 muscarinic acetylcholine, adrenocorticotropin, endothelin-A, and thyrotropin-releasing hormone (11Peralta E. Winslow J. Peterson G. Smith D. Ashkenazi A. Ramachandran J. Scmerlik M. Capon D. Science. 1987; 236: 600-605Crossref PubMed Scopus (333) Google Scholar, 12Naville D. Jaillard C. Barjhoux L. Durand P. Begeot M. Biochem. Biophys. Res. Commun. 1997; 230: 7-12Crossref PubMed Scopus (47) Google Scholar, 13Hosoda K. Nakao K. Tamura N. Arai H. Ogawa Y. Suga S. Nakanishi S. Imura H. J. Biol. Chem. 1992; 267: 18797-18804Abstract Full Text PDF PubMed Google Scholar, 14Iwasaki T. Yamada M. Satoh T. Konaka S. Ren Y. Hashimoto K. Kohga H. Kato Y. Mori M. J. Biol. Chem. 1996; 271: 22183-22188Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar).Because primer extension analysis could not detect the expression of the hGHS-R gene in the pituitary as expected by its extremely low expression (4Howard A.D. Feighner S.D. Cully D.F. Arena J.P. Liberator P.A. Rosenblum C.I. Hamelin M. Hreniuk D.L. Palyha O.C. Anderson J. Paress P.S. Diaz C. Chou M. Liu K.K. McKee K.K. Pong S-S. Chaung L-Y.P. Elbrecht A. Dashkevicz M. Heavens R. Rigby M. Sirinathsinghji D.J.S. Dean D.C. Mellilo D.G. Patchett A.A. Nargund R.P. Griffin P.R. Demartino J.A. Guputa S.K. Schaeffer J.M. Smith R.G. Van der Ploeg L.H.T. Science. 1996; 273: 974-977Crossref PubMed Scopus (1787) Google Scholar, 6Guan X-M., Yu,H. Palyha O.C. Mckee K.K. Feighner S.D. Sirinathsinghji D.J.S. Smith R.G. Van der Ploeg L.H.T. Howard A.D. Mol. Brain Res. 1997; 48: 23-29Crossref PubMed Scopus (971) Google Scholar, 9Bennett P.A. Thomas G.B. Howard A.D. Feighner S.D. Van der Ploeg L.H.T. Smith R.G. Robinson I.C.A.F. Endocrinology. 1997; 138: 4552-4557Crossref PubMed Scopus (168) Google Scholar), 5′-RACE analysis was conducted to determine the transcription initiation site. The direct sequencing of the 5′-RACE products suggests that the major 5′-end of the hGHS-R cDNA is −453 bp relative to the translation initiation site. However, we obtained at least 13 RACE products with different 5′-termini. Thus, we cannot exclude the possibility that the hGHS-R gene has multiple initiation sites like other G protein-coupled receptors such as those for the dopamine D4 (15Kamakura S. Iwaki A. Matsumoto M. Fukumaki Y. Biochem. Biophys. Res. Commun. 1997; 235: 321-326Crossref PubMed Scopus (48) Google Scholar), thyroid-stimulating hormone (16Ikuyama S. Niller H.H. Shimura H. Akamizu T. Kohn L.D. Mol. Endocrinol. 1992; 6: 793-804Crossref PubMed Scopus (46) Google Scholar), α1B-adrenergic hormone (17Ramarao C.S. Denker J.M. Perez D.M. Gavin R.J. Riek R.P. Graham R.M. J. Biol. Chem. 1992; 267: 21936-21945Abstract Full Text PDF PubMed Google Scholar), β1-adrenergic hormone (18Searles R.P. Midson C.N. Nipper V.J. Machida C.A. J. Biol. Chem. 1995; 270: 157-162Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), and luteinizing hormone (LH) (19Wang H. Nelson S. Ascoli M. Segaloff D.L. Mol. Endocrinol. 1992; 6: 320-326PubMed Google Scholar). Sequence analysis of the 5′-untranslated flanking region of the hGHS-R gene indicated that the region upstream of the putative transcriptional start site does not have any of the typical characteristics of promoter regions such as a TATA box, CAAT box, or the GC-rich region. The TATA-less promoter, found primarily in housekeeping genes, is characteristic of G protein-coupled receptors such as those for β-adrenergic hormone, α1B-adrenergic hormone, dopamine D4, LH, thyroid-stimulating hormone, gonadotropin-releasing hormone, thyrotropin-releasing hormone, and GH-releasing hormone (13Hosoda K. Nakao K. Tamura N. Arai H. Ogawa Y. Suga S. Nakanishi S. Imura H. J. Biol. Chem. 1992; 267: 18797-18804Abstract Full Text PDF PubMed Google Scholar, 14Iwasaki T. Yamada M. Satoh T. Konaka S. Ren Y. Hashimoto K. Kohga H. Kato Y. Mori M. J. Biol. Chem. 1996; 271: 22183-22188Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 15Kamakura S. Iwaki A. Matsumoto M. Fukumaki Y. Biochem. Biophys. Res. Commun. 1997; 235: 321-326Crossref PubMed Scopus (48) Google Scholar, 16Ikuyama S. Niller H.H. Shimura H. Akamizu T. Kohn L.D. Mol. Endocrinol. 1992; 6: 793-804Crossref PubMed Scopus (46) Google Scholar, 17Ramarao C.S. Denker J.M. Perez D.M. Gavin R.J. Riek R.P. Graham R.M. J. Biol. Chem. 1992; 267: 21936-21945Abstract Full Text PDF PubMed Google Scholar, 18Searles R.P. Midson C.N. Nipper V.J. Machida C.A. J. Biol. Chem. 1995; 270: 157-162Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 19Wang H. Nelson S. Ascoli M. Segaloff D.L. Mol. Endocrinol. 1992; 6: 320-326PubMed Google Scholar, 20Albarracin C.T. Kaiser U.B. Chin W.W. Endocrinology. 1994; 135: 2300-2306Crossref PubMed Scopus (75) Google Scholar, 21Fan N.C. Peng C. Krisinger J. Leung P.C.K. Mol. Cell. Endocrinol. 1995; 107: R1-R8Crossref PubMed Scopus (88) Google Scholar, 22Tsai-Morris C.H. Xie X. Wang W. Buczko E. Dufau M.L. J. Biol. Chem. 1993; 268: 4447-4452Abstract Full Text PDF PubMed Google Scholar, 23Heckert L.L. Daley I.J. Griswold M.D. Mol. Endocrinol. 1992; 6: 70-80PubMed Google Scholar, 24Petersenn S. Rasch A.C. Heynes M. Schulte H.M. Mol. Endocrinol. 1998; 12: 233-247Crossref PubMed Google Scholar). As there is a TdT-Inr family-like element that overlaps with the putative transcription start site, initiator binding protein other than TATA-binding protein may be a rate-limiting factor for the hGHS-R gene transcription initiation.In addition to the Inr-like sequence, there are several putative sites for binding transcription factors as shown in Fig. 2. Of particular interest is the presence of consensus sequences for the POU-domain transcription factors, Pit-1, Oct-1, and Ptx1, which have been shown to be involved in pituitary-specific expression (25Bonder M. Karin M. Cell. 1987; 50: 267-275Abstract Full Text PDF PubMed Scopus (209) Google Scholar, 26Elsholtz H.P. Albert V.R. Treacy M.N. Rosenfeld M.G. Genes Dev. 1990; 4: 43-51Crossref PubMed Scopus (62) Google Scholar, 27Tremblay J.J. Lanctot C. Drouin J. Mol. Endocrinol. 1998; 12: 428-441Crossref PubMed Google Scholar). The octamer binding protein Oct-1 is expressed ubiquitously and activates certain eukaryotic TATA-less promoters (26Elsholtz H.P. Albert V.R. Treacy M.N. Rosenfeld M.G. Genes Dev. 1990; 4: 43-51Crossref PubMed Scopus (62) Google Scholar). Ptx1 is expressed in all pituitary cell types and is essential for transcription of the glycoprotein hormone α-subunit gene and Lim3/hk3 and activates transcription of the GH or PRL gene in synergy with Pit-1 in somatolactotrophs, the LHβ gene with SF-1 in gonadotrophs (27Tremblay J.J. Lanctot C. Drouin J. Mol. Endocrinol. 1998; 12: 428-441Crossref PubMed Google Scholar), and the POMC gene with NeuroD1, one of bHLH factors, in corticotrophs (28Poulin G. Turgeon B. Drouin J. Mol. Cell. Biol. 1997; 17: 6673-6682Crossref PubMed Google Scholar). Putative binding sites for several transcription factors, such as bHLH factors and AP2, were also identified on the hGHS-R gene, some of which may be responsible for the basal activity of the hGHS-R gene promoter. The presence of multiple putative sites for AP2 binding suggests their involvement in mediating transcriptional activation by phorbol esters and cAMP (29Imagawa M. Chiu R. Karin M. Cell. 1987; 51: 251-260Abstract Full Text PDF PubMed Scopus (1029) Google Scholar). There were no GH response elements such as the signal transducers and activators of transcription (STAT)3 and STAT5, cis-inducible elements, or serum-response elements. The reported inhibition of rat GHS-R mRNA expression in the hypothalamus by GH (9Bennett P.A. Thomas G.B. Howard A.D. Feighner S.D. Van der Ploeg L.H.T. Smith R.G. Robinson I.C.A.F. Endocrinology. 1997; 138: 4552-4557Crossref PubMed Scopus (168) Google Scholar) may be caused by an indirect action of GH.The promoter activity of the hGHS-R 5′-flanking region was assessed after insertion upstream of the luciferase coding sequence in the pGL3-Basic vector. The activity of the GHS-R promoter was detected in GH3 cells but not in HeLa human epithelioid carcinoma of cervix cells and EP1 human neuroblastoma cells, suggesting that this DNA fragment is necessary and sufficient to drive expression of a heterologous gene in GH3. This finding is consistent with the recent report demonstrating the expression of GHS-R mRNA in GH3 cells (8Adams E.F. Huang B. Buchfelder M. Howard A. Smith R.G. Feighner S.D. Van der Pleog L.H.T. Bowers C.Y. Fahlbush R. J. Clin. Endocrinol. Metab. 1998; 83: 638-642PubMed Google Scholar). Deletion studies allowed us to define positive regulatory elements for the basal promoter activity located between −734 and −608, where there are putative binding sites for Pit-1, PEA-3, AP2, and Ptx1. However, the small decrease of promoter activity by deleting from −734 to −669, where two Pit-1 binding like elements located, did not support strong Pit-1 dependence. Furthermore, deletion of the upstream fragments from −1224 to −734 led to a significant increase in the promoter activity, suggesting that this region works as a negative regulatory element such as a repressor in the hGHS-R gene. Further study is required to determine which transcription factors play a key role in the hGHS-R gene transcription.In summary, the 5′-flanking region of the hGHS-R gene contains a TATA-less promoter with cell-specific activity and putative binding sites for several transcription factors in the regions required for the basal activity. These initial characterizations should facilitate further study of the mechanisms involved in the transcriptional regulation of the hGHS-R gene expression in human health and disease. Growth hormone (GH)1 secretion is regulated mainly by the hypothalamic stimulatory factor, GH-releasing hormone, and the inhibitory factor, somatostatin. On the other hand, GH secretagogues have been developed as a small synthetic peptide, GH-releasing peptide (1Momany F.A. Bowers C.Y. Reynolds G.A. Hong A. Newlander K. Endocrinology. 1984; 114: 1531-1536Crossref PubMed Scopus (175) Google Scholar), and non-peptides, L-692,429 (2Smith R.G. Chen K. Schoen W.R. Pong S-S. Hickey G.J. Jacks T.M. Butler B.S. Chan W.W-S. Chaung L-Y.P. Judith F. Taylor A.M. Wyvratt Jr., M.J. Fisher M.H. Science. 1993; 260: 1640-1643Crossref PubMed Scopus (327) Google Scholar) and MK-0677 (3Patchett A.A. Nargund R.P. Tata J.R. Chen M-H. Barakat K.J. Johnston D.B.R. Chen K. Chan W.W-S. Butler B.S. Hickey G.J. Jacks T.M. Scleim K. Pong S-S. Chaung L-Y.P. Chen H.Y. Fraizier E. Leung K.H. Chui S.-H.L. Smith R.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7001-7005Crossref PubMed Scopus (337) Google Scholar), with potent GH-secreting activity, especially in vivo and in humans. The recent cloning of the human, porcine (4Howard A.D. Feighner S.D. Cully D.F. Arena J.P. Liberator P.A. Rosenblum C.I. Hamelin M. Hreniuk D.L. Palyha O.C. Anderson J. Paress P.S. Diaz C. Chou M. Liu K.K. McKee K.K. Pong S-S. Chaung L-Y.P. Elbrecht A. Dashkevicz M. Heavens R. Rigby M. Sirinathsinghji D.J.S. Dean D.C. Mellilo D.G. Patchett A.A. Nargund R.P. Griffin P.R. Demartino J.A. Guputa S.K. Schaeffer J.M. Smith R.G. Van der Ploeg L.H.T. Science. 1996; 273: 974-977Crossref PubMed Scopus (1787) Google Scholar), and rat (5McKee K.K. Palyha O.C. Feighner S.D. Hreniuk D.L. Tan C.P. Phillips M.S. Smith R.G. Van der Ploeg L.H.T. Howard A.D. Mol. Endocrinol. 1997; 11: 415-423Crossref PubMed Scopus (289) Google Scholar) GHS receptor (GHS-R) cDNA has suggested an additional physiological regulation for GH release. GHS-R mRNA is expressed not only in the pituitary and hypothalamus but also in the hippocampus, pancreas (6Guan X-M., Yu,H. Palyha O.C. Mckee K.K. Feighner S.D. Sirinathsinghji D.J.S. Smith R.G. Van der Ploeg L.H.T. Howard A.D. Mol. Brain Res. 1997; 48: 23-29Crossref PubMed Scopus (971) Google Scholar), and neuroendocrine tumors (7de Keyzer Y. Lenne F. Bertagna X. Eur. J. Endocrinol. 1997; 137: 715-718Crossref PubMed Scopus (50) Google Scholar), including human somatotropinomas and rat GH3 cells (8Adams E.F. Huang B. Buchfelder M. Howard A. Smith R.G. Feighner S.D. Van der Pleog L.H.T. Bowers C.Y. Fahlbush R. J. Clin. Endocrinol. Metab. 1998; 83: 638-642PubMed Google Scholar). There are still only a few reports about the regulation of GHS-R expression. Bennettet al. (9Bennett P.A. Thomas G.B. Howard A.D. Feighner S.D. Van der Ploeg L.H.T. Smith R.G. Robinson I.C.A.F. Endocrinology. 1997; 138: 4552-4557Crossref PubMed Scopus (168) Google Scholar) have recently reported that GHS-R expression in the hypothalamus was markedly increased in dw/dw dwarf rats and was down-regulated in dw/dw rats treated with GH. They have also reported that GHS-R mRNA expression in the ventromedial nucleus of the hypothalamus was lower in male than in female rats. To understand the transcriptional regulation of the human GHS-R (hGHS-R) gene expression, we have cloned and characterized the 5′-flanking region of the hGHS-R gene. DISCUSSIONThe present study is the first report of a cloning and characterization of a human genomic DNA fragment containing the 5′-flanking region of the hGHS-R gene. When the sequence of the 5′-flanking region of the hGHS-R gene was compared with the upstream sequence of the hGHS-R cDNA, a single exon upstream of the coding exon was identified as shown for other human G protein-coupled receptor genes such as those for the M2 muscarinic acetylcholine, adrenocorticotropin, endothelin-A, and thyrotropin-releasing hormone (11Peralta E. Winslow J. Peterson G. Smith D. Ashkenazi A. Ramachandran J. Scmerlik M. Capon D. Science. 1987; 236: 600-605Crossref PubMed Scopus (333) Google Scholar, 12Naville D. Jaillard C. Barjhoux L. Durand P. Begeot M. Biochem. Biophys. Res. Commun. 1997; 230: 7-12Crossref PubMed Scopus (47) Google Scholar, 13Hosoda K. Nakao K. Tamura N. Arai H. Ogawa Y. Suga S. Nakanishi S. Imura H. J. Biol. Chem. 1992; 267: 18797-18804Abstract Full Text PDF PubMed Google Scholar, 14Iwasaki T. Yamada M. Satoh T. Konaka S. Ren Y. Hashimoto K. Kohga H. Kato Y. Mori M. J. Biol. Chem. 1996; 271: 22183-22188Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar).Because primer extension analysis could not detect the expression of the hGHS-R gene in the pituitary as expected by its extremely low expression (4Howard A.D. Feighner S.D. Cully D.F. Arena J.P. Liberator P.A. Rosenblum C.I. Hamelin M. Hreniuk D.L. Palyha O.C. Anderson J. Paress P.S. Diaz C. Chou M. Liu K.K. McKee K.K. Pong S-S. Chaung L-Y.P. Elbrecht A. Dashkevicz M. Heavens R. Rigby M. Sirinathsinghji D.J.S. Dean D.C. Mellilo D.G. Patchett A.A. Nargund R.P. Griffin P.R. Demartino J.A. Guputa S.K. Schaeffer J.M. Smith R.G. Van der Ploeg L.H.T. Science. 1996; 273: 974-977Crossref PubMed Scopus (1787) Google Scholar, 6Guan X-M., Yu,H. Palyha O.C. Mckee K.K. Feighner S.D. Sirinathsinghji D.J.S. Smith R.G. Van der Ploeg L.H.T. Howard A.D. Mol. Brain Res. 1997; 48: 23-29Crossref PubMed Scopus (971) Google Scholar, 9Bennett P.A. Thomas G.B. Howard A.D. Feighner S.D. Van der Ploeg L.H.T. Smith R.G. Robinson I.C.A.F. Endocrinology. 1997; 138: 4552-4557Crossref PubMed Scopus (168) Google Scholar), 5′-RACE analysis was conducted to determine the transcription initiation site. The direct sequencing of the 5′-RACE products suggests that the major 5′-end of the hGHS-R cDNA is −453 bp relative to the translation initiation site. However, we obtained at least 13 RACE products with different 5′-termini. Thus, we cannot exclude the possibility that the hGHS-R gene has multiple initiation sites like other G protein-coupled receptors such as those for the dopamine D4 (15Kamakura S. Iwaki A. Matsumoto M. Fukumaki Y. Biochem. Biophys. Res. Commun. 1997; 235: 321-326Crossref PubMed Scopus (48) Google Scholar), thyroid-stimulating hormone (16Ikuyama S. Niller H.H. Shimura H. Akamizu T. Kohn L.D. Mol. Endocrinol. 1992; 6: 793-804Crossref PubMed Scopus (46) Google Scholar), α1B-adrenergic hormone (17Ramarao C.S. Denker J.M. Perez D.M. Gavin R.J. Riek R.P. Graham R.M. J. Biol. Chem. 1992; 267: 21936-21945Abstract Full Text PDF PubMed Google Scholar), β1-adrenergic hormone (18Searles R.P. Midson C.N. Nipper V.J. Machida C.A. J. Biol. Chem. 1995; 270: 157-162Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), and luteinizing hormone (LH) (19Wang H. Nelson S. Ascoli M. Segaloff D.L. Mol. Endocrinol. 1992; 6: 320-326PubMed Google Scholar). Sequence analysis of the 5′-untranslated flanking region of the hGHS-R gene indicated that the region upstream of the putative transcriptional start site does not have any of the typical characteristics of promoter regions such as a TATA box, CAAT box, or the GC-rich region. The TATA-less promoter, found primarily in housekeeping genes, is characteristic of G protein-coupled receptors such as those for β-adrenergic hormone, α1B-adrenergic hormone, dopamine D4, LH, thyroid-stimulating hormone, gonadotropin-releasing hormone, thyrotropin-releasing hormone, and GH-releasing hormone (13Hosoda K. Nakao K. Tamura N. Arai H. Ogawa Y. Suga S. Nakanishi S. Imura H. J. Biol. Chem. 1992; 267: 18797-18804Abstract Full Text PDF PubMed Google Scholar, 14Iwasaki T. Yamada M. Satoh T. Konaka S. Ren Y. Hashimoto K. Kohga H. Kato Y. Mori M. J. Biol. Chem. 1996; 271: 22183-22188Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 15Kamakura S. Iwaki A. Matsumoto M. Fukumaki Y. Biochem. Biophys. Res. Commun. 1997; 235: 321-326Crossref PubMed Scopus (48) Google Scholar, 16Ikuyama S. Niller H.H. Shimura H. Akamizu T. Kohn L.D. Mol. Endocrinol. 1992; 6: 793-804Crossref PubMed Scopus (46) Google Scholar, 17Ramarao C.S. Denker J.M. Perez D.M. Gavin R.J. Riek R.P. Graham R.M. J. Biol. Chem. 1992; 267: 21936-21945Abstract Full Text PDF PubMed Google Scholar, 18Searles R.P. Midson C.N. Nipper V.J. Machida C.A. J. Biol. Chem. 1995; 270: 157-162Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 19Wang H. Nelson S. Ascoli M. Segaloff D.L. Mol. Endocrinol. 1992; 6: 320-326PubMed Google Scholar, 20Albarracin C.T. Kaiser U.B. Chin W.W. Endocrinology. 1994; 135: 2300-2306Crossref PubMed Scopus (75) Google Scholar, 21Fan N.C. Peng C. Krisinger J. Leung P.C.K. Mol. Cell. Endocrinol. 1995; 107: R1-R8Crossref PubMed Scopus (88) Google Scholar, 22Tsai-Morris C.H. Xie X. Wang W. Buczko E. Dufau M.L. J. Biol. Chem. 1993; 268: 4447-4452Abstract Full Text PDF PubMed Google Scholar, 23Heckert L.L. Daley I.J. Griswold M.D. Mol. Endocrinol. 1992; 6: 70-80PubMed Google Scholar, 24Petersenn S. Rasch A.C. Heynes M. Schulte H.M. Mol. Endocrinol. 1998; 12: 233-247Crossref PubMed Google Scholar). As there is a TdT-Inr family-like element that overlaps with the putative transcription start site, initiator binding protein other than TATA-binding protein may be a rate-limiting factor for the hGHS-R gene transcription initiation.In addition to the Inr-like sequence, there are several putative sites for binding transcription factors as shown in Fig. 2. Of particular interest is the presence of consensus sequences for the POU-domain transcription factors, Pit-1, Oct-1, and Ptx1, which have been shown to be involved in pituitary-specific expression (25Bonder M. Karin M. Cell. 1987; 50: 267-275Abstract Full Text PDF PubMed Scopus (209) Google Scholar, 26Elsholtz H.P. Albert V.R. Treacy M.N. Rosenfeld M.G. Genes Dev. 1990; 4: 43-51Crossref PubMed Scopus (62) Google Scholar, 27Tremblay J.J. Lanctot C. Drouin J. Mol. Endocrinol. 1998; 12: 428-441Crossref PubMed Google Scholar). The octamer binding protein Oct-1 is expressed ubiquitously and activates certain eukaryotic TATA-less promoters (26Elsholtz H.P. Albert V.R. Treacy M.N. Rosenfeld M.G. Genes Dev. 1990; 4: 43-51Crossref PubMed Scopus (62) Google Scholar). Ptx1 is expressed in all pituitary cell types and is essential for transcription of the glycoprotein hormone α-subunit gene and Lim3/hk3 and activates transcription of the GH or PRL gene in synergy with Pit-1 in somatolactotrophs, the LHβ gene with SF-1 in gonadotrophs (27Tremblay J.J. Lanctot C. Drouin J. Mol. Endocrinol. 1998; 12: 428-441Crossref PubMed Google Scholar), and the POMC gene with NeuroD1, one of bHLH factors, in corticotrophs (28Poulin G. Turgeon B. Drouin J. Mol. Cell. Biol. 1997; 17: 6673-6682Crossref PubMed Google Scholar). Putative binding sites for several transcription factors, such as bHLH factors and AP2, were also identified on the hGHS-R gene, some of which may be responsible for the basal activity of the hGHS-R gene promoter. The presence of multiple putative sites for AP2 binding suggests their involvement in mediating transcriptional activation by phorbol esters and cAMP (29Imagawa M. Chiu R. Karin M. Cell. 1987; 51: 251-260Abstract Full Text PDF PubMed Scopus (1029) Google Scholar). There were no GH response elements such as the signal transducers and activators of transcription (STAT)3 and STAT5, cis-inducible elements, or serum-response elements. The reported inhibition of rat GHS-R mRNA expression in the hypothalamus by GH (9Bennett P.A. Thomas G.B. Howard A.D. Feighner S.D. Van der Ploeg L.H.T. Smith R.G. Robinson I.C.A.F. Endocrinology. 1997; 138: 4552-4557Crossref PubMed Scopus (168) Google Scholar) may be caused by an indirect action of GH.The promoter activity of the hGHS-R 5′-flanking region was assessed after insertion upstream of the luciferase coding sequence in the pGL3-Basic vector. The activity of the GHS-R promoter was detected in GH3 cells but not in HeLa human epithelioid carcinoma of cervix cells and EP1 human neuroblastoma cells, suggesting that this DNA fragment is necessary and sufficient to drive expression of a heterologous gene in GH3. This finding is consistent with the recent report demonstrating the expression of GHS-R mRNA in GH3 cells (8Adams E.F. Huang B. Buchfelder M. Howard A. Smith R.G. Feighner S.D. Van der Pleog L.H.T. Bowers C.Y. Fahlbush R. J. Clin. Endocrinol. Metab. 1998; 83: 638-642PubMed Google Scholar). Deletion studies allowed us to define positive regulatory elements for the basal promoter activity located between −734 and −608, where there are putative binding sites for Pit-1, PEA-3, AP2, and Ptx1. However, the small decrease of promoter activity by deleting from −734 to −669, where two Pit-1 binding like elements located, did not support strong Pit-1 dependence. Furthermore, deletion of the upstream fragments from −1224 to −734 led to a significant increase in the promoter activity, suggesting that this region works as a negative regulatory element such as a repressor in the hGHS-R gene. Further study is required to determine which transcription factors play a key role in the hGHS-R gene transcription.In summary, the 5′-flanking region of the hGHS-R gene contains a TATA-less promoter with cell-specific activity and putative binding sites for several transcription factors in the regions required for the basal activity. These initial characterizations should facilitate further study of the mechanisms involved in the transcriptional regulation of the hGHS-R gene expression in human health and disease. The present study is the first report of a cloning and characterization of a human genomic DNA fragment containing the 5′-flanking region of the hGHS-R gene. When the sequence of the 5′-flanking region of the hGHS-R gene was compared with the upstream sequence of the hGHS-R cDNA, a single exon upstream of the coding exon was identified as shown for other human G protein-coupled receptor genes such as those for the M2 muscarinic acetylcholine, adrenocorticotropin, endothelin-A, and thyrotropin-releasing hormone (11Peralta E. Winslow J. Peterson G. Smith D. Ashkenazi A. Ramachandran J. Scmerlik M. Capon D. Science. 1987; 236: 600-605Crossref PubMed Scopus (333) Google Scholar, 12Naville D. Jaillard C. Barjhoux L. Durand P. Begeot M. Biochem. Biophys. Res. Commun. 1997; 230: 7-12Crossref PubMed Scopus (47) Google Scholar, 13Hosoda K. Nakao K. Tamura N. Arai H. Ogawa Y. Suga S. Nakanishi S. Imura H. J. Biol. Chem. 1992; 267: 18797-18804Abstract Full Text PDF PubMed Google Scholar, 14Iwasaki T. Yamada M. Satoh T. Konaka S. Ren Y. Hashimoto K. Kohga H. Kato Y. Mori M. J. Biol. Chem. 1996; 271: 22183-22188Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Because primer extension analysis could not detect the expression of the hGHS-R gene in the pituitary as expected by its extremely low expression (4Howard A.D. Feighner S.D. Cully D.F. Arena J.P. Liberator P.A. Rosenblum C.I. Hamelin M. Hreniuk D.L. Palyha O.C. Anderson J. Paress P.S. Diaz C. Chou M. Liu K.K. McKee K.K. Pong S-S. Chaung L-Y.P. Elbrecht A. Dashkevicz M. Heavens R. Rigby M. Sirinathsinghji D.J.S. Dean D.C. Mellilo D.G. Patchett A.A. Nargund R.P. Griffin P.R. Demartino J.A. Guputa S.K. Schaeffer J.M. Smith R.G. Van der Ploeg L.H.T. Science. 1996; 273: 974-977Crossref PubMed Scopus (1787) Google Scholar, 6Guan X-M., Yu,H. Palyha O.C. Mckee K.K. Feighner S.D. Sirinathsinghji D.J.S. Smith R.G. Van der Ploeg L.H.T. Howard A.D. Mol. Brain Res. 1997; 48: 23-29Crossref PubMed Scopus (971) Google Scholar, 9Bennett P.A. Thomas G.B. Howard A.D. Feighner S.D. Van der Ploeg L.H.T. Smith R.G. Robinson I.C.A.F. Endocrinology. 1997; 138: 4552-4557Crossref PubMed Scopus (168) Google Scholar), 5′-RACE analysis was conducted to determine the transcription initiation site. The direct sequencing of the 5′-RACE products suggests that the major 5′-end of the hGHS-R cDNA is −453 bp relative to the translation initiation site. However, we obtained at least 13 RACE products with different 5′-termini. Thus, we cannot exclude the possibility that the hGHS-R gene has multiple initiation sites like other G protein-coupled receptors such as those for the dopamine D4 (15Kamakura S. Iwaki A. Matsumoto M. Fukumaki Y. Biochem. Biophys. Res. Commun. 1997; 235: 321-326Crossref PubMed Scopus (48) Google Scholar), thyroid-stimulating hormone (16Ikuyama S. Niller H.H. Shimura H. Akamizu T. Kohn L.D. Mol. Endocrinol. 1992; 6: 793-804Crossref PubMed Scopus (46) Google Scholar), α1B-adrenergic hormone (17Ramarao C.S. Denker J.M. Perez D.M. Gavin R.J. Riek R.P. Graham R.M. J. Biol. Chem. 1992; 267: 21936-21945Abstract Full Text PDF PubMed Google Scholar), β1-adrenergic hormone (18Searles R.P. Midson C.N. Nipper V.J. Machida C.A. J. Biol. Chem. 1995; 270: 157-162Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), and luteinizing hormone (LH) (19Wang H. Nelson S. Ascoli M. Segaloff D.L. Mol. Endocrinol. 1992; 6: 320-326PubMed Google Scholar). Sequence analysis of the 5′-untranslated flanking region of the hGHS-R gene indicated that the region upstream of the putative transcriptional start site does not have any of the typical characteristics of promoter regions such as a TATA box, CAAT box, or the GC-rich region. The TATA-less promoter, found primarily in housekeeping genes, is characteristic of G protein-coupled receptors such as those for β-adrenergic hormone, α1B-adrenergic hormone, dopamine D4, LH, thyroid-stimulating hormone, gonadotropin-releasing hormone, thyrotropin-releasing hormone, and GH-releasing hormone (13Hosoda K. Nakao K. Tamura N. Arai H. Ogawa Y. Suga S. Nakanishi S. Imura H. J. Biol. Chem. 1992; 267: 18797-18804Abstract Full Text PDF PubMed Google Scholar, 14Iwasaki T. Yamada M. Satoh T. Konaka S. Ren Y. Hashimoto K. Kohga H. Kato Y. Mori M. J. Biol. Chem. 1996; 271: 22183-22188Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 15Kamakura S. Iwaki A. Matsumoto M. Fukumaki Y. Biochem. Biophys. Res. Commun. 1997; 235: 321-326Crossref PubMed Scopus (48) Google Scholar, 16Ikuyama S. Niller H.H. Shimura H. Akamizu T. Kohn L.D. Mol. Endocrinol. 1992; 6: 793-804Crossref PubMed Scopus (46) Google Scholar, 17Ramarao C.S. Denker J.M. Perez D.M. Gavin R.J. Riek R.P. Graham R.M. J. Biol. Chem. 1992; 267: 21936-21945Abstract Full Text PDF PubMed Google Scholar, 18Searles R.P. Midson C.N. Nipper V.J. Machida C.A. J. Biol. Chem. 1995; 270: 157-162Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 19Wang H. Nelson S. Ascoli M. Segaloff D.L. Mol. Endocrinol. 1992; 6: 320-326PubMed Google Scholar, 20Albarracin C.T. Kaiser U.B. Chin W.W. Endocrinology. 1994; 135: 2300-2306Crossref PubMed Scopus (75) Google Scholar, 21Fan N.C. Peng C. Krisinger J. Leung P.C.K. Mol. Cell. Endocrinol. 1995; 107: R1-R8Crossref PubMed Scopus (88) Google Scholar, 22Tsai-Morris C.H. Xie X. Wang W. Buczko E. Dufau M.L. J. Biol. Chem. 1993; 268: 4447-4452Abstract Full Text PDF PubMed Google Scholar, 23Heckert L.L. Daley I.J. Griswold M.D. Mol. Endocrinol. 1992; 6: 70-80PubMed Google Scholar, 24Petersenn S. Rasch A.C. Heynes M. Schulte H.M. Mol. Endocrinol. 1998; 12: 233-247Crossref PubMed Google Scholar). As there is a TdT-Inr family-like element that overlaps with the putative transcription start site, initiator binding protein other than TATA-binding protein may be a rate-limiting factor for the hGHS-R gene transcription initiation. In addition to the Inr-like sequence, there are several putative sites for binding transcription factors as shown in Fig. 2. Of particular interest is the presence of consensus sequences for the POU-domain transcription factors, Pit-1, Oct-1, and Ptx1, which have been shown to be involved in pituitary-specific expression (25Bonder M. Karin M. Cell. 1987; 50: 267-275Abstract Full Text PDF PubMed Scopus (209) Google Scholar, 26Elsholtz H.P. Albert V.R. Treacy M.N. Rosenfeld M.G. Genes Dev. 1990; 4: 43-51Crossref PubMed Scopus (62) Google Scholar, 27Tremblay J.J. Lanctot C. Drouin J. Mol. Endocrinol. 1998; 12: 428-441Crossref PubMed Google Scholar). The octamer binding protein Oct-1 is expressed ubiquitously and activates certain eukaryotic TATA-less promoters (26Elsholtz H.P. Albert V.R. Treacy M.N. Rosenfeld M.G. Genes Dev. 1990; 4: 43-51Crossref PubMed Scopus (62) Google Scholar). Ptx1 is expressed in all pituitary cell types and is essential for transcription of the glycoprotein hormone α-subunit gene and Lim3/hk3 and activates transcription of the GH or PRL gene in synergy with Pit-1 in somatolactotrophs, the LHβ gene with SF-1 in gonadotrophs (27Tremblay J.J. Lanctot C. Drouin J. Mol. Endocrinol. 1998; 12: 428-441Crossref PubMed Google Scholar), and the POMC gene with NeuroD1, one of bHLH factors, in corticotrophs (28Poulin G. Turgeon B. Drouin J. Mol. Cell. Biol. 1997; 17: 6673-6682Crossref PubMed Google Scholar). Putative binding sites for several transcription factors, such as bHLH factors and AP2, were also identified on the hGHS-R gene, some of which may be responsible for the basal activity of the hGHS-R gene promoter. The presence of multiple putative sites for AP2 binding suggests their involvement in mediating transcriptional activation by phorbol esters and cAMP (29Imagawa M. Chiu R. Karin M. Cell. 1987; 51: 251-260Abstract Full Text PDF PubMed Scopus (1029) Google Scholar). There were no GH response elements such as the signal transducers and activators of transcription (STAT)3 and STAT5, cis-inducible elements, or serum-response elements. The reported inhibition of rat GHS-R mRNA expression in the hypothalamus by GH (9Bennett P.A. Thomas G.B. Howard A.D. Feighner S.D. Van der Ploeg L.H.T. Smith R.G. Robinson I.C.A.F. Endocrinology. 1997; 138: 4552-4557Crossref PubMed Scopus (168) Google Scholar) may be caused by an indirect action of GH. The promoter activity of the hGHS-R 5′-flanking region was assessed after insertion upstream of the luciferase coding sequence in the pGL3-Basic vector. The activity of the GHS-R promoter was detected in GH3 cells but not in HeLa human epithelioid carcinoma of cervix cells and EP1 human neuroblastoma cells, suggesting that this DNA fragment is necessary and sufficient to drive expression of a heterologous gene in GH3. This finding is consistent with the recent report demonstrating the expression of GHS-R mRNA in GH3 cells (8Adams E.F. Huang B. Buchfelder M. Howard A. Smith R.G. Feighner S.D. Van der Pleog L.H.T. Bowers C.Y. Fahlbush R. J. Clin. Endocrinol. Metab. 1998; 83: 638-642PubMed Google Scholar). Deletion studies allowed us to define positive regulatory elements for the basal promoter activity located between −734 and −608, where there are putative binding sites for Pit-1, PEA-3, AP2, and Ptx1. However, the small decrease of promoter activity by deleting from −734 to −669, where two Pit-1 binding like elements located, did not support strong Pit-1 dependence. Furthermore, deletion of the upstream fragments from −1224 to −734 led to a significant increase in the promoter activity, suggesting that this region works as a negative regulatory element such as a repressor in the hGHS-R gene. Further study is required to determine which transcription factors play a key role in the hGHS-R gene transcription. In summary, the 5′-flanking region of the hGHS-R gene contains a TATA-less promoter with cell-specific activity and putative binding sites for several transcription factors in the regions required for the basal activity. These initial characterizations should facilitate further study of the mechanisms involved in the transcriptional regulation of the hGHS-R gene expression in human health and disease. We thank Chikako Ogata for excellent technical assistance.

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