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

Abstract

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 that the of the hGHS-R cDNA is −453 to the translation initiation site. we with we the that the hGHS-R gene has initiation sites other G protein-coupled such as those for the S. A. M. Y. Biochem. Biophys. Res. Commun. 1997; PubMed Scopus Google Scholar), hormone S. H. T. Mol. Endocrinol. 1992; PubMed Scopus Google Scholar), hormone J.M. R.P. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar), hormone R.P. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), and luteinizing hormone H. S. M. D.L. Mol. Endocrinol. 1992; Google Scholar). analysis of the region of the hGHS-R gene that the region upstream of the putative transcriptional start site not have of the typical of promoter such as a or the The in is of G protein-coupled such as those for hormone, hormone, hormone, hormone, thyrotropin-releasing hormone, and GH-releasing hormone 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, S. A. M. Y. Biochem. Biophys. Res. Commun. 1997; PubMed Scopus Google Scholar, S. H. T. Mol. Endocrinol. 1992; PubMed Scopus Google Scholar, J.M. R.P. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar, R.P. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, H. S. M. D.L. Mol. Endocrinol. 1992; Google Scholar, Endocrinology. PubMed Scopus Google Scholar, C. J. Leung Mol. Endocrinol. 1995; PubMed Scopus Google Scholar, X. E. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, Mol. Endocrinol. 1992; Google Scholar, S. M. Mol. Endocrinol. 1998; PubMed Google Scholar). is a that with the putative transcription start initiator binding other than a for the hGHS-R gene transcription to the are several putative sites for binding transcription factors as shown in is the of for the transcription and have been shown to in expression M. M. 1987; Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, C. J. Mol. Endocrinol. 1998; PubMed Google Scholar). The binding is expressed and PubMed Scopus Google Scholar). is expressed in pituitary and is for transcription of the hormone gene and and transcription of the GH or gene in with in the gene with in C. J. Mol. Endocrinol. 1998; PubMed Google Scholar), and the gene with of in G. B. J. Mol. Biol. 1997; PubMed Google Scholar). binding sites for several transcription such as factors and also identified the hGHS-R of for the activity of the hGHS-R gene The of putative sites for binding in transcriptional by and M. R. M. 1987; Full Text PDF PubMed Scopus Google Scholar). There no GH such as the signal transducers and activators of transcription and or The reported 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) by an of promoter activity of the hGHS-R 5′-flanking region was upstream of the luciferase coding sequence in the The activity of the GHS-R promoter was in GH3 cells but not in HeLa human of cells and EP1 human that DNA fragment is and to expression of a gene in is with the recent report 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). to for the promoter activity −734 and are putative binding sites for and the small of promoter activity by from −734 to binding not deletion of the upstream from −1224 to −734 to a in the promoter activity, that region as a such as a in the hGHS-R gene. study is to determine transcription factors a in the hGHS-R gene the 5′-flanking region of the hGHS-R gene a promoter with activity and putative binding sites for several transcription factors in the for the facilitate further study of the in the transcriptional regulation of the hGHS-R gene expression in human and 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 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. 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 that the of the hGHS-R cDNA is −453 to the translation initiation site. we with we the that the hGHS-R gene has initiation sites other G protein-coupled such as those for the S. A. M. Y. Biochem. Biophys. Res. Commun. 1997; PubMed Scopus Google Scholar), hormone S. H. T. Mol. Endocrinol. 1992; PubMed Scopus Google Scholar), hormone J.M. R.P. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar), hormone R.P. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), and luteinizing hormone H. S. M. D.L. Mol. Endocrinol. 1992; Google Scholar). analysis of the region of the hGHS-R gene that the region upstream of the putative transcriptional start site not have of the typical of promoter such as a or the The in is of G protein-coupled such as those for hormone, hormone, hormone, hormone, thyrotropin-releasing hormone, and GH-releasing hormone 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, S. A. M. Y. Biochem. Biophys. Res. Commun. 1997; PubMed Scopus Google Scholar, S. H. T. Mol. Endocrinol. 1992; PubMed Scopus Google Scholar, J.M. R.P. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar, R.P. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, H. S. M. D.L. Mol. Endocrinol. 1992; Google Scholar, Endocrinology. PubMed Scopus Google Scholar, C. J. Leung Mol. Endocrinol. 1995; PubMed Scopus Google Scholar, X. E. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, Mol. Endocrinol. 1992; Google Scholar, S. M. Mol. Endocrinol. 1998; PubMed Google Scholar). is a that with the putative transcription start initiator binding other than a for the hGHS-R gene transcription to the are several putative sites for binding transcription factors as shown in is the of for the transcription and have been shown to in expression M. M. 1987; Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, C. J. Mol. Endocrinol. 1998; PubMed Google Scholar). The binding is expressed and PubMed Scopus Google Scholar). is expressed in pituitary and is for transcription of the hormone gene and and transcription of the GH or gene in with in the gene with in C. J. Mol. Endocrinol. 1998; PubMed Google Scholar), and the gene with of in G. B. J. Mol. Biol. 1997; PubMed Google Scholar). binding sites for several transcription such as factors and also identified the hGHS-R of for the activity of the hGHS-R gene The of putative sites for binding in transcriptional by and M. R. M. 1987; Full Text PDF PubMed Scopus Google Scholar). There no GH such as the signal transducers and activators of transcription and or The reported 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) by an of promoter activity of the hGHS-R 5′-flanking region was upstream of the luciferase coding sequence in the The activity of the GHS-R promoter was in GH3 cells but not in HeLa human of cells and EP1 human that DNA fragment is and to expression of a gene in is with the recent report 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). to for the promoter activity −734 and are putative binding sites for and the small of promoter activity by from −734 to binding not deletion of the upstream from −1224 to −734 to a in the promoter activity, that region as a such as a in the hGHS-R gene. study is to determine transcription factors a in the hGHS-R gene the 5′-flanking region of the hGHS-R gene a promoter with activity and putative binding sites for several transcription factors in the for the facilitate further study of the in the transcriptional regulation of the hGHS-R gene expression in human and 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). 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 that the of the hGHS-R cDNA is −453 to the translation initiation site. we with we the that the hGHS-R gene has initiation sites other G protein-coupled such as those for the S. A. M. Y. Biochem. Biophys. Res. Commun. 1997; PubMed Scopus Google Scholar), hormone S. H. T. Mol. Endocrinol. 1992; PubMed Scopus Google Scholar), hormone J.M. R.P. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar), hormone R.P. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), and luteinizing hormone H. S. M. D.L. Mol. Endocrinol. 1992; Google Scholar). analysis of the region of the hGHS-R gene that the region upstream of the putative transcriptional start site not have of the typical of promoter such as a or the The in is of G protein-coupled such as those for hormone, hormone, hormone, hormone, thyrotropin-releasing hormone, and GH-releasing hormone 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, S. A. M. Y. Biochem. Biophys. Res. Commun. 1997; PubMed Scopus Google Scholar, S. H. T. Mol. Endocrinol. 1992; PubMed Scopus Google Scholar, J.M. R.P. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar, R.P. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, H. S. M. D.L. Mol. Endocrinol. 1992; Google Scholar, Endocrinology. PubMed Scopus Google Scholar, C. J. Leung Mol. Endocrinol. 1995; PubMed Scopus Google Scholar, X. E. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, Mol. Endocrinol. 1992; Google Scholar, S. M. Mol. Endocrinol. 1998; PubMed Google Scholar). is a that with the putative transcription start initiator binding other than a for the hGHS-R gene transcription to the are several putative sites for binding transcription factors as shown in is the of for the transcription and have been shown to in expression M. M. 1987; Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, C. J. Mol. Endocrinol. 1998; PubMed Google Scholar). The binding is expressed and PubMed Scopus Google Scholar). is expressed in pituitary and is for transcription of the hormone gene and and transcription of the GH or gene in with in the gene with in C. J. Mol. Endocrinol. 1998; PubMed Google Scholar), and the gene with of in G. B. J. Mol. Biol. 1997; PubMed Google Scholar). binding sites for several transcription such as factors and also identified the hGHS-R of for the activity of the hGHS-R gene The of putative sites for binding in transcriptional by and M. R. M. 1987; Full Text PDF PubMed Scopus Google Scholar). There no GH such as the signal transducers and activators of transcription and or The reported 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) by an of GH. The promoter activity of the hGHS-R 5′-flanking region was upstream of the luciferase coding sequence in the The activity of the GHS-R promoter was in GH3 cells but not in HeLa human of cells and EP1 human that DNA fragment is and to expression of a gene in is with the recent report 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). to for the promoter activity −734 and are putative binding sites for and the small of promoter activity by from −734 to binding not deletion of the upstream from −1224 to −734 to a in the promoter activity, that region as a such as a in the hGHS-R gene. study is to determine transcription factors a in the hGHS-R gene transcription. the 5′-flanking region of the hGHS-R gene a promoter with activity and putative binding sites for several transcription factors in the for the facilitate further study of the in the transcriptional regulation of the hGHS-R gene expression in human and for