Albarracin C T, Kaiser U B, Chin W W
Department of Medicine, Brigham and Women's Hospital, Howard Hughes Medical Institute, Boston, Massachusetts 02115.
Endocrinology. 1994 Dec;135(6):2300-6. doi: 10.1210/endo.135.6.7988412.
The GnRH receptor (GnRH-R) is a cell surface, G protein-coupled receptor that is highly expressed in pituitary gonadotropes. Activation of the receptor by GnRH stimulates the release of FSH and LH. Pituitary GnRH-R numbers and, hence, gonadotrope responsiveness to GnRH vary under different conditions and are regulated to a large extent by GnRH itself. To study the transcriptional regulation of the GnRH-R gene, a genomic clone containing 1.2 kilobases (kb) of the 5'-flanking region of mouse GnRH-R gene was isolated and characterized. A major transcriptional start site was identified 62 nucleotides upstream of the translational start site by primer extension and ribonuclease protection analyses. The promoter region does not contain canonical TATA sequences in the appropriate location. To determine whether this putative promoter is functional, it was subcloned into a luciferase reporter plasmid (GnRH-RLuc), and its transient expression was studied in cell lines of gonadotrope (alpha T3-1) and somatolactotrope (GH3) origins as well as those of nonpituitary origin (JEG-3 and CV-1). Luciferase activity was increased in alpha T3-1 (246-fold +/- 34.5-fold; P < 0.005) compared with the promoterless vector control but was considerably lower in GH3 (41-fold +/- 3.9-fold; P < 0.005), JEG-3 (12-fold +/- 0.9-fold; P < 0.005) and CV-1 (8-fold +/- 1.3-fold) indicating that GnRH-RLuc is preferentially expressed in cells of gonadotrope origin. Furthermore, GnRH agonist stimulated luciferase activity 3.4-fold +/- 0.3-fold (P < 0.005) above basal levels in GH3 cells cotransfected with rat GnRH-R complementary DNA, indicating that the GnRH-R promoter sequence is responsive to this ligand. In summary, we have identified and partially characterized the promoter region of the mouse GnRH-R and demonstrated that the regulatory elements for tissue-specific expression as well as for GnRH regulation are present within a 1.2-kb 5'-flanking region of the mouse GnRH-R gene.
促性腺激素释放激素受体（GnRH-R）是一种细胞表面的G蛋白偶联受体，在垂体促性腺细胞中高度表达。GnRH激活该受体可刺激促卵泡激素（FSH）和促黄体生成素（LH）的释放。垂体GnRH-R的数量，进而促性腺细胞对GnRH的反应性，在不同条件下会有所变化，并且在很大程度上受GnRH自身调节。为了研究GnRH-R基因的转录调控，分离并鉴定了一个包含小鼠GnRH-R基因5'侧翼区1.2千碱基（kb）的基因组克隆。通过引物延伸和核糖核酸酶保护分析，在翻译起始位点上游62个核苷酸处确定了一个主要转录起始位点。启动子区域在合适位置不包含典型的TATA序列。为了确定这个假定的启动子是否具有功能，将其亚克隆到荧光素酶报告质粒（GnRH-RLuc）中，并在促性腺细胞系（αT3-1）、生长激素催乳素细胞系（GH3）以及非垂体来源的细胞系（JEG-3和CV-1）中研究其瞬时表达。与无启动子载体对照相比，αT3-1细胞中的荧光素酶活性增加（246倍±34.5倍；P<0.005），但在GH3细胞（41倍±3.9倍；P<0.005）、JEG-3细胞（12倍±0.9倍；P<0.005）和CV-1细胞（8倍±1.3倍）中则显著较低，这表明GnRH-RLuc在促性腺细胞来源的细胞中优先表达。此外，在与大鼠GnRH-R互补DNA共转染的GH3细胞中，GnRH激动剂刺激荧光素酶活性比基础水平高出3.4倍±0.3倍（P<0.005），这表明GnRH-R启动子序列对该配体有反应。总之，我们已经鉴定并部分表征了小鼠GnRH-R的启动子区域，并证明小鼠GnRH-R基因1.2-kb的5'侧翼区内存在组织特异性表达以及GnRH调节的调控元件。
