A BINDING-SITE FOR THE CYCLIC ADENOSINE 3',5'-MONOPHOSPHATE-RESPONSE ELEMENT-BINDING PROTEIN AS A REGULATORY ELEMENT IN THE GRP78-PROMOTER
A BINDING-SITE FOR THE CYCLIC ADENOSINE 3',5'-MONOPHOSPHATE-RESPONSE ELEMENT-BINDING PROTEIN AS A REGULATORY ELEMENT IN THE GRP78-PROMOTER
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DOI：
10.1210/mend-5-12-1862
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发表时间：
1991-12-01
影响因子：
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通讯作者：
LEE, AS
中科院分区：
文献类型：
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作者：
ALEXANDRE, S;NAKAKI, T;LEE, AS
78 kda葡萄糖调节蛋白（GRP78）在许多细胞类型中普遍表达。它的启动子包含多个蛋白质结合位点和功能元件。在这项研究中，我们使用b淋巴细胞和HeLa细胞的核提取物检测了大鼠grp78启动子的高亲和力蛋白结合位点bp -198至-180。该区域包含一个序列TGACGTGA，除一个碱基外，该序列与cAMP-response element （CRE）相同。位点定向突变表明，该序列在仓鼠成纤维细胞中是一个主要的基础水平调控元件，也是在应激诱导条件下维持高启动子活性所必需的。通过凝胶迁移位移分析，我们检测到两种特定的蛋白质复合物。主要的特异性复合物I虽然在免疫学上不同于42 kda的CRE结合蛋白（CREB），但它与grp位点的结合最强烈，但也与CRE一致序列具有亲和力。因此，复合体I可能由CREB/激活转录因子蛋白家族的其他成员组成。次要特异性复合体II由CREB或与其抗原相关的蛋白质组成。非特异性复合物III由Ku自身抗原组成，这是HeLa核提取物中丰富的70至80 kda蛋白复合物。通过共转染实验，我们证明在F9畸胎癌细胞中，grp78启动子可以被磷酸化的CREB或当CREB转染的细胞用钙离子载体A23187处理时被反式激活。讨论了cAMP在特定细胞类型和组织中对grp78基因的差异调控。
The 78-kDa glucose-regulated protein (GRP78) is ubiquitously expressed in many cell types. Its promoter contains multiple protein-binding sites and functional elements. In this study we examined a high affinity protein-binding site spanning bp -198 to -180 of the rat grp78 promoter, using nuclear extracts from both B-lymphoid and HeLa cells. This region contains a sequence TGACGTGA which, with the exception of one base, is identical to the cAMP-response element (CRE). Site-directed mutagenesis reveals that this sequence functions as a major basal level regulatory element in hamster fibroblast cells and is also necessary to maintain high promoter activity under stress-induced conditions. By gel mobility shift analysis, we detect two specific protein complexes. The major specific complex I, while immunologically distinct from the 42-kDa CRE-binding protein (CREB), binds most strongly to the grp site, but also exhibits affinity for the CRE consensus sequence. As such, complex I may consist of other members of the CREB/activating transcription factor protein family. The minor specific complex II consists of CREB or a protein antigenically related to it. A nonspecific complex III consists of the Ku autoantigen, an abundant 70- to 80-kDa protein complex in HeLa nuclear extracts. By cotransfection experiments, we demonstrate that in F9 teratocarcinoma cells, the grp78 promoter can be trans-activated by the phosphorylated CREB or when the CREB-transfected cells are treated with the calcium ionophore A23187. The differential regulation of the grp78 gene by cAMP in specific cell types and tissues is discussed.