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

Abstract

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 ؊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 cotransfected 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. Growth hormone-releasing hormone (GHRH)1 plays a major role in stimulating both synthesis and release of growth hormone (GH) in the anterior pituitary through its specific receptor. So far, the GHRH receptor (GHRH-R) has been cloned in human (1, 2), mouse (3), rat (1), and pig (4). The GHRH-R is a member of G protein-coupled receptor family and transduces GHRH-dependent increase in intracellular cAMP via G s activation for stimulating somatotroph proliferation and GH gene expression (1-4). Lin et al. (5) demonstrated that one amino acid substitution of the GHRH-R in the little mouse, showing genetically transmitted dwarfism, caused GH deficiency and somatotroph hypoplasia. In addition, an amber-type mutation (Glu72Stop) of the GHRH-R in humans was demonstrated to cause profound GH deficiency (6). These genetic disorders suggest the physiological significance of GHRH-R in hypothalamopituitary GH axis.The relationship between the dynamics of GHRH-R expression and GH secretion remains to be clarified. Not only the functional defects of GHRH-R but also the amount of GHRH-R should affect GH synthesis and secretion in the pituitary. Hypothalamic hormone, neurotransmitters, various hormonal states, and nutrition all could modulate the activity of the GHRH-GH axis. For instance, glucocorticoids potentiate GHRH action and enhance GH secretion in rats (7-9), where...

The role of pit-1 in the regulation of the rat growth hormone-releasing hormone receptor gene transcription by glucocorticoids

“…2 that the transcription from dGpG with no pit-1 site responded to DEX by 4·4-fold (Fig. 2), while it coincides well with previous results that showed that the transcription of the GHRH-receptor gene requires pit-1 (Lin et al 1992, Iguchi et al 1999, Miller et al 1999, Nogami et al 2002. The only explanation possible for this discrepancy is that some DNA sequences within dGpG-p that are absent in dGpG, the pit-1 site and the 14-base pair spacer between pGRE and the pit-1 site, contain the silencer element.…”

“…The PCR fragment obtained by primer A and one of these was placed upstream of the tk promoter to generate dGpGmp or dGpG-pm respectively. Similarly, the primers carrying pit-1 sequences of the rat GH gene (rGH-2, Andersen & Rosenfeld 1994, 5 -CTGATGG ATAATTTAAAGGATGGTGGGACATT-3 ), the rat prolactin gene (rPrl-1P, Andersen & Rosenfeld 1994, 5 -CATGAATATATATATAAATGGTGGGACATT-3 ) and the human GHRH-receptor gene (hGHRHR-P2, Iguchi et al 1999, 5 -CGCTGAATATTCACCAGGAT GGTGGGACATT-3 ) were used to generate dGpGGHp, dGpG-PRLp and dGpG-hGHRHRp respectively (see Fig. 6).…”

“…In a previous study, we identified two GREs, a thyroid hormone response element (TRE) and a binding site for pit-1, a pituitary specific transcription factor (Bodner et al 1988, Ingraham et al 1988, in the upstream region of the rat GHRH-receptor gene (Nogami et al 2002). Pit-1 plays a principal role in the cell type specification of prolactin (PRL), growth hormone (GH) and a subset of thyroid stimulating hormone (TSH) cells, and is a prerequisite for the transcriptional activation of the genes that are specifically expressed in these cells (Bodner et al 1988, Ingraham et al 1988, including GHRH-receptor gene (Lin et al 1992, Iguchi et al 1999, Miller et al 1999, Nogami et al 2002. However, pit-1 alone may not be able to induce expression of pituitary-specific genes.…”

International Union of Pharmacology. XXXV. The Glucagon Receptor Family

“…There is no clear correspondence between exons of the gene and functional domains of the receptor protein. Functional analysis of the gene promoter in cell transfection assays reveals a selective preference for expression in pituitary cells (Iguchi et al, 1999;Miller et al, 1999) likely due to the presence of the transcription factor Pit-1, which positively regulates GHRH receptor gene expression (Lin et al, 1992;Iguchi et al, 1999;Miller et al, 1999;Salvatori et al, 2002). Glucocorticoid regulation has also been mapped to a composite regula- FIG.…”

Novel Function of the Transactivation Domain of a Pituitary-specific Transcription Factor, Pit-1

“…It is involved in the development of the three pituitary cell types, somatotrophs, lactotrophs, and thyrotrophs, as well as the gene expression of growth hormone (GH), 1 prolactin (PRL), GH-releasing hormone receptor (GHRH-R), thyrotropin ␤ subunit (TSH ␤), and Pit-1 itself by binding to the specific DNA elements of these genes (3)(4)(5)(6)(7)(8)(9)(10). Therefore, abnormalities of the Pit-1 gene result in GH, PRL, and TSH deficiencies.…”