Analysis of regulatory mechanism for gene exprssions of growth hormone-releasing peptide receptor and growth hormone-releasing hormone receptor

生长激素释放肽受体及生长激素释放激素受体基因表达调控机制分析

• 批准号: 09671059
• 负责人: OKIMURA Yasuhiko
• 金额: $ 1.98万
• 依托单位: Kobe University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671059/
• 关键词: Growth Hormone; Growth hormon-releasing peptide receptor; Growth hormon-releasing hormone receptor; pituitary; Pit-1; gene expression

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 kb upstream from the translation start site. RNase protection analysis showed the major transcription start site is 122bp upstream from the translation start site. The 5'-end of the longest initial 5'-RACE product was close to the site. There were no typical TATA homologies but several putative regulatory elements including Pit-i binding site-like element. Transient transfection studies using a luciferase reporter gene demonstrated 5-flanking region had promoter activity in GH3 cells (derived from rat pituitary tumor) but not in non-pituitary cells, BeWo and HeLa cells. However, co-transfection of Pit-1 expression vector increased luciferase activity in BeWo cells. Deletion study showed 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 less contributied to the … More 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 elements, P1 and P2, located from -129 to -123 and from -171 to - 160, respectively. Both mobility shift assay and DNase-l foot print analysis showed that P2 had much higher Pit -1 binding affinity than P1. These findings were consistent with the results that the region from -310 to - 130 is an important element for Pit-i-dependent expression of GHRH-R.In addition, we cloned human growth hormone secretagogue receptor (GHS-R) gene containing the 5-flanking region of 0.6-2.9 kb. Analysis of the hGHS-R transcripts with 5'RACE suggested that putative transcription initiation site was -453 bp upstream from the translation start site. There was no TATA, CAAT, or GC box but an initiator-like sequence sequence. The 5'-flanking region was inserted into a luciferase reporter vector had promoter activity in GH3 cells. The hGHS-R promoter activity appeared to be from -734 to -608. Less

我们克隆了人生长激素释放激素受体(GHRH-R)基因的5‘侧翼区，并测定了其上游2.7kb的核苷酸序列。核糖核酸酶保护分析表明，主要转录起始点位于翻译起始点上游122bp。最长的初始5‘-RACE产物的5’-末端靠近现场。没有典型的TATA同源序列，但有几个可能的调控元件，包括Pit-I结合位点样元件。使用荧光素酶报告基因的瞬时转基因研究表明，5-侧翼区在GH3细胞(来源于大鼠垂体瘤)中具有启动子活性，但在非垂体细胞、BeWo和HeLa细胞中不具有启动子活性。然而，共转染Pit-1表达载体提高了BeWo细胞中荧光素酶的活性。缺失研究表明，-310到-130和-130到-120区域对生长激素释放激素受体基因的表达很重要，尽管后者对…的贡献较小更多的基因表达。在共转染Pit-1表达载体的BeWo细胞中，-310~-130区域是依赖Pit-1表达GHRH-R基因所必需的。从-310到-120的区域有两个可能的Pit-1结合元件P1和P2，分别位于-129到-123和-171到-160之间。迁移率改变分析和DNA酶-L足迹分析均表明，P2与Pit-1的结合亲和力明显高于P1。这些结果与以下结果相一致，即-310-130区域是GHRH-R依赖于Pit-I表达的重要元件。此外，我们还克隆了人生长激素促分泌素受体(GHS-R)基因，其5-侧翼区为0.6-2.9kb。对带有5‘RACE的hGHS-R转录本的分析表明，推测的转录起始点位于翻译起始点上游-453bp。没有TATA、CAAT或GC盒，只有类似启动子的序列。5‘侧翼区被插入到荧光素酶报告载体中，在GH3细胞中具有启动子活性。HGHS-R启动子活性在-734~-608之间。较少

项目成果

Kaji H.: "Cloning and characterization of the 5'-flanking region of the human growth hormone secretagogue receptor gene." J Biol. Chem.273・51. 33885-33888 (1998)

Kaji H.：“人类生长激素促分泌素受体基因 5 侧翼区域的克隆和表征”，J Biol.273·51（1998）。

Iida K.: "Growth hormone(GH)insensitirity synclrome with high serum GH-binding pretein levels caused by a heterozygous splice site mutation of the GH receptor gene producing a bsck of inlra ullulan donain." J.Clin.Erdocrinol.Metrb.83. 531-537 (1998)

Iida K.：“生长激素 (GH) 不敏感综合症，伴有高血清 GH 结合蛋白水平，这是由 GH 受体基因的杂合剪接位点突变引起的，产生了内 ullulan doain 的 bsck。”

Iguchi G: "Cloning and characterization of the 5'-flanking region of the human growth hormone releasing hormone receptor gene" J Biol Chem. (in press). (1999)

Iguchi G：“人类生长激素释放激素受体基因 5 侧翼区域的克隆和表征”J Biol Chem。

Nowakowski BE: "Characterization of DNA regions mediating the ability of Ca^<2+>/Calmodulindependcnt protein kinase II to seimulate prolactin promoter actirity." Mol.Cell.Endouinol.132. 109-116 (1997)

Nowakowski BE：“介导 Ca^2/钙调蛋白依赖性蛋白激酶 II 模拟催乳素启动子活性能力的 DNA 区域的表征。”

Kaji H: "Cloning and characterization of the 5'-flanking region of the human growth hormone secretagogue receptor gene" J Biol Chem. 273 (51). 33885-33888 (1998)

Kaji H：“人类生长激素促分泌素受体基因 5 侧翼区域的克隆和表征”J Biol Chem。