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Study of the role of EGF high-affinity binding site and the signal transduction in the transfectants of A-431 cell.

A-431细胞转染子中EGF高亲和力结合位点的作用及其信号转导的研究。

基本信息

批准号：
04671131
负责人：
ATSUMI Toshiko
金额：
$ 1.09万
依托单位：
Meikai University, School of Dentistry
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04671131/
关键词：
EGF rounding high-affinity binding site PKC PKA ラウンディング

项目摘要

EGF is known to pull the trigger of signal transduction of growth. Using A-341 cells and their transfectants. we studied the relation between EGF high-affinity binding sites and rounding induced by EGF and the roles of protein kinase C (PKC) and protein kinase A (PKA) in EGF-induced cell rounding.Elevation of intracellular Ca^<2+> concentration ([Ca^<2+>]_1) and rounding took place when A-431 cells were sitimulated with EGF(10^<-8>M), Pretreatment with 12-0-tetradecanoyl-phorbol-13-acetate (TPA, 100ng/ml), an activator of PKC, for 30 min inhibited rounding, elevation of [Ca^<2+>] _1, and expression of high-affinity binding sites for EGF.But, when pretreatment with TPA(100ng/ml) was carry out for 20 h, which treatment is known to result in down-regulation of PKC, all three of the above responses to EGF occured. Preteatment with forskolin (100mu M), an activator of PKA, for 30 min gave the same result as that with TPA (100ng/ml) for 20 h. On the other hand, in the case of the An-10 cells, lacking high-affinity binding sites for EGF, stimulated with EGF (10^<-8>M) did not kead to rounding, but elevationof [Ca^<2+>] _i did occur. Pretreatment of TPA (100ng/ml) for 30 min inhibited rounding, elevationof [Ca^<2+>] _i and expression of high-affinity binding sites for EGF same as in A-431 cells. But, pretreatment of the cells with TPA (100ng/ml) for 20 h caused cell rounding, elevationof [Ca^<2+>] _i and expression of high-affinity binding sites for EGF.However rounding and high-affinity binding sites were not found in the case of no pretreat ment of Am-10 cells. Forskolin (100mu M) pretreatment for 30 min gave about the same results as TPA (100ng/ml) pretreatment for 20 hours.these results suggest that founding of A-431 cells induced by EGF was injibited by activation of PKC bia PLC, but accelerated by activation of PKA.Furthermoer, the rounding response apearsto correlate with the presense of high-affinity binding sites for EGF.

EGF是一种促进生长的信号转导因子。使用A-341细胞及其转染子。我们研究了EGF高亲和力结合位点与EGF诱导的细胞变圆的关系以及蛋白激酶C（PKC）和蛋白激酶A（PKA）在EGF诱导的细胞变圆中的作用，发现EGF（10 μ M）刺激A-431细胞后，细胞内Ca^2+浓度（[Ca^2+] 1）升高，细胞变圆<-8>;用12-0-十四酰基-佛波醇-13-乙酸酯预处理PKC激活剂TPA（100 ng/ml）作用30 min，可抑制细胞变圆、[Ca^2+] _1升高及EGF高亲和力结合位点的表达，但对EGF高亲和力结合位点的表达无明显影响。当用TPA（100 ng/ml）预处理20小时（已知该处理导致PKC下调）时，对EGF的上述三种反应均发生。用PKA激活剂Forskolin（100 μ M）预处理30分钟，与TPA（100 ng/ml）预处理20小时结果相同。另一方面，缺乏EGF高亲和力结合位点的An-10细胞，经EGF（10 μ <-8>M）刺激后，细胞内[Ca^（2+）] _i升高，但不引起细胞变圆。TPA（100 ng/ml）预处理30 min可抑制A-431细胞变圆、[Ca^<2+>] _i升高和EGF高亲和力结合位点的表达。TPA（100 ng/ml）预处理20 h可使细胞变圆，[Ca^<2+>] _i升高，EGF高亲和力结合位点表达，而未预处理的Am-10细胞未发现细胞变圆和EGF高亲和力结合位点表达。Forskolin（100 μ M）预处理30分钟与TPA（100 ng/ml）预处理20小时的结果基本相同，提示EGF诱导的A-431细胞的形成受PLC激活PKC的抑制，而PKA的激活则加速了EGF诱导的A-431细胞的形成，并提示EGF诱导的A-431细胞的形成与EGF高亲和力结合位点的存在有关。