Cloning and characterization of the 5 '-flanking region of the human growth hormone-releasing hormone receptor gene

We cloned the 5'-flanking region of the human growth hormone-releasing horm one receptor (GHRH-R) gene and determined the nucleotide sequence of 2.7 ki lobases 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 a mplification of cDNA ends was close to the site. There were no typical TATA homologies but several putative regulatory elements including Pit-1-bindin g site-like element. Transient transfection studies using a luciferase repo rter gene demonstrated that 5'-flanking region had promoter activity in GH3 cells (derived from rat pituitary tumor) but not in nonpituitary cells, Be Wo and HeLa cells. However, co-transfection of Pit-1 expression vector incr eased luciferase activity in BeWo cells. Deletion study showed that the reg ions from -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 g ene expression. In BeWo cells cotransfected with Pit-1 expression vector, t he region from -310 to -130 was essential for the Pit-1-dependent expressio n of GHRH-R gene. The region from -310 to -120 has two putative Pit-1-bindi ng sites, P1 and P2, located from -129 to -123 and from -171 to -160, respe ctively. Both mobility shift assay and DNase-I footprint analysis showed th at P2 had much higher Pit-1 binding affinity than P1. Mutation of P2 decrea sed GHRH-R gene expression in GH3 cells. These findings were consistent wit h the results that the region from -310 to -130 is an important element for Pit-1-dependent expression of GHRH-R gene.