Molecular organization of the mouse gastrin-releasing peptide receptor gene and its promoter.
Molecular organization of the mouse gastrin-releasing peptide receptor gene and its promoter.
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小鼠胃泌素释放肽受体基因及其启动子的分子结构。
DOI：
10.1016/s0378-1119(99)00563-6
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发表时间：
2000
期刊：
影响因子：
3.5
通讯作者：
Battey,JF
中科院分区：
文献类型：
--
作者：
Weber,HC;Jensen,RT;Battey,JF
小鼠胃泌素释放肽受体(mGRP-R)是G蛋白偶联受体家族的一员，介导其特异性配体胃肠激素/神经递质GRP的重要生理作用，包括对小鼠瑞士3T3成纤维细胞的促有丝分裂作用。糖皮质激素和细胞内cAMP的增加被报道改变GRP-R基因的转录，但这些影响的分子基础尚不清楚。为了开始确定影响mGRP-R表达的可能的基因调控机制，我们确定了它的结构，并研究了它的基本启动子活性。我们分离并鉴定了编码小鼠胃泌素释放肽受体(mGRP-R)的基因组噬菌体P1克隆。通过DNA测序和Southern印迹分析，我们确定了蛋白质编码区包含在三个外显子中，其中包括两个内含子20和2kb。GRP-R基因的开放阅读框编码一个384个氨基酸的蛋白，与小鼠BRS-3蛋白的同源性为48%，与小鼠NMB-R蛋白的同源性为53%。利用5‘非编码区的小卫星多态和荧光原位杂交技术，将mGRP-R基因定位于X染色体(DXMit20)。在天然表达mGRP-R的瑞士3T3细胞中，Northern印迹分析可检测到3kb和7kb两种基因特异性的mRNA。通过核糖核酸酶保护实验，并独立地通过5‘RACE克隆的反向PCR，发现共同的mRNA起始点聚集在TTTAAA基序下游21~61bp之间，该基序位于ATG翻译起始点上游450bp处。然而，使用了不同的聚腺苷酸化位点。将2kb的基因组DNA片段从2147~141个碱基5‘端克隆到荧光素酶报告基因表达载体中，在瑞士3T3和COS-7细胞中表达。启动子的渐进性截断和位于TTTAAA基序上游83bp的环状AMP反应元件(CRE)的突变表明，只有当TTTAAA基序和完整的CRE位点都被保留时，才能在瑞士3T3细胞中发生转录上的mGRP-R激活。随着本研究报道的mGRP-R基因的完整结构和最小启动子序列的获得，将有可能在未来的研究中探讨糖皮质激素、cAMP等因素对mGRP-R基因转录调控的分子基础。
The murine gastrin-releasing peptide receptor (mGRP-R) is a member of the G protein-coupled receptor family and mediates important physiological actions of its specific ligand, the gastrointestinal hormone/neurotransmitter GRP, including mitogenic properties in the mouse Swiss 3T3 fibroblasts. Glucocorticoids and increases in intracellular cAMP are reported to alter GRP-R gene transcription, but the molecular basis for these effects is unknown. To begin to identify possible gene regulatory mechanisms that are responsible for modifying mGRP-R expression, we determined its structure and investigated its basal promoter activity. We isolated and characterized genomic bacteriophage P1 clones encoding the mouse gastrin-releasing peptide receptor (mGRP-R). By DNA sequencing and Southern blot analyses, we determined the protein coding region to be contained in three exons interrupted by two introns 20 and 2kb in length. The open reading frame of the putative GRP-R gene encodes for a 384-amino-acid protein which demonstrates 48% identity with the mouse BRS-3 protein and 53% identity with the mouse NMB-R protein. The mGRP-R gene locus extends over 29kb and was mapped to the X-chromosome (DXMit20) utilizing a minisatellite polymorphism in the 5′ UTR and by fluorescent in-situ hybridization (FISH). In Swiss 3T3 cells, which natively express mGRP-R, two gene-specific mRNA species of 3 and 7kb can be detected by Northern blot analysis. With RNase protection assays, and independently with inverse PCR of 5′ RACE clones, common mRNA initiation sites were identified clustered between 21 and 61bp downstream of a TTTAAA motif, which is located 450bp upstream of the ATG translation start site. However, different polyadenylation sites are utilized. A 2kb genomic DNA fragment extending from 2147 to 141 bases 5′ to the ATG translation start was cloned into a luciferase reporter plasmid and shown to contain promoter activity in Swiss 3T3 and COS-7 cells. Progressive promoter truncations and mutations of a cyclic AMP response element (CRE) located 83bp upstream of the TTTAAA motif demonstrate that transcriptional mGRP-R activation in Swiss 3T3 cells only occurs when both the TTTAAA motif and the intact CRE site are retained. With the availability of the full structure of the mGRP-R gene and the minimal promoter sequences reported in this study, it will be possible in future studies to investigate the molecular basis for transcriptional regulation of the mGRP-R gene by glucocorticoids, cAMP and other factors.