Oetjen E, Diedrich T, Eggers A, Eckert B, Knepel W
Department of Biochemical Pharmacology, University of Göttingen, Germany.
J Biol Chem. 1994 Oct 28;269(43):27036-44.
The cAMP response element (CRE)-binding transcription factor CREB can mediate induction of gene transcription in response to calcium as well as to cAMP. Since the rat insulin I gene 5'-flanking region contains a CRE with an octamer-like motif (TGACGTCC), CREB binding and cAMP/calcium responsiveness of the insulin CRE were investigated. In an electrophoretic mobility shift assay and in Southwestern blot experiments, bacterially expressed recombinant CREB bound to the insulin CRE as it did to the rat glucagon and rat somatostatin gene CREs. However, in nuclear extracts of the pancreatic islet cell line HIT, protein complexes binding to the insulin CRE did not contain proteins with CREB-like immunoreactivity, although these bound to the glucagon and somatostatin CREs. When reporter fusion genes were transfected into HIT cells, the isolated insulin CRE increased basal activity and mediated transcriptional activation by cAMP. However, cAMP stimulation of transcription through the insulin CRE was weak when compared with the response through the glucagon and somatostatin CREs. Furthermore, the insulin CRE did not confer responsiveness to membrane depolarization and calcium influx, in contrast to the glucagon and somatostatin CREs. These results demonstrate that the functional properties of the rat insulin I gene CRE are different from those of the rat glucagon and somatostatin CREs which may be explained by a distinct pattern of nuclear protein binding and suggest the existence of post-translational mechanisms that decrease the binding of cellular CREB to the insulin CRE.
环磷酸腺苷（cAMP）反应元件（CRE）结合转录因子CREB可介导基因转录的诱导，以响应钙以及cAMP。由于大鼠胰岛素I基因5'-侧翼区含有一个带有八聚体样基序（TGACGTCC）的CRE，因此对胰岛素CRE的CREB结合及cAMP/钙反应性进行了研究。在电泳迁移率变动分析和蛋白质印迹实验中，细菌表达的重组CREB与胰岛素CRE结合，就如同它与大鼠胰高血糖素和大鼠生长抑素基因的CRE结合一样。然而，在胰岛细胞系HIT的核提取物中，与胰岛素CRE结合的蛋白质复合物并不包含具有CREB样免疫反应性的蛋白质，尽管这些蛋白质与胰高血糖素和生长抑素的CRE结合。当将报告基因融合基因转染到HIT细胞中时，分离出的胰岛素CRE增加了基础活性，并介导了cAMP的转录激活。然而，与通过胰高血糖素和生长抑素的CRE的反应相比，通过胰岛素CRE的cAMP转录刺激较弱。此外，与胰高血糖素和生长抑素的CRE不同，胰岛素CRE并未赋予对膜去极化和钙内流的反应性。这些结果表明，大鼠胰岛素I基因CRE的功能特性与大鼠胰高血糖素和生长抑素的CRE不同，这可能由核蛋白结合的独特模式来解释，并提示存在翻译后机制，该机制可减少细胞CREB与胰岛素CRE的结合。
