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J Biol Chem, Vol. 274, Issue 17, 12108-12114, April 23, 1999
From the Third Division, Department of Medicine, Kobe University
School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku,
Kobe 650-0017, Japan
We cloned the 5'-flanking region of the human
growth hormone-releasing hormone receptor (GHRH-R) gene and determined
the nucleotide sequence of 2.7 kilobases upstream from the translation
start site. RNase protection analysis showed the major transcription start site is 122 base pairs upstream from the translation start site.
The 5'-end of the longest product of 5'-rapid amplification of cDNA
ends was close to the site. There were no typical TATA homologies but
several putative regulatory elements including Pit-1-binding site-like
element. Transient transfection studies using a luciferase reporter
gene demonstrated that 5'-flanking region had promoter activity in GH3
cells (derived from rat pituitary tumor) but not in nonpituitary cells,
BeWo and HeLa cells. However, co-transfection of Pit-1 expression
vector increased luciferase activity in BeWo cells. Deletion study
showed that the regions from
Cloning and Characterization of the 5'-Flanking Region of the
Human Growth Hormone-releasing Hormone Receptor Gene
310 to 130 and from 130 to 120
were important for the GHRH-R gene expression in GH3 cells, although
the latter contributed less to the gene expression. In BeWo cells
co-transfected with Pit-1 expression vector, the region from 310 to
130 was essential for the Pit-1-dependent expression of
GHRH-R gene. The region from 310 to 120 has two putative
Pit-1-binding sites, P1 and P2, located from 129 to 123 and from
171 to 160, respectively. Both mobility shift assay and DNase-I
footprint analysis showed that P2 had much higher Pit-1 binding
affinity than P1. Mutation of P2 decreased GHRH-R gene expression in
GH3 cells. These findings were consistent with the results that the
region from 310 to 130 is an important element for
Pit-1-dependent expression of GHRH-R gene.
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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