GPⅡb^(R995A)点突变对受体亲合力及PP125^(FAK)磷酸化的影响

Effects of Point Mutation in Glycoprotein Ⅱ b^(R995A) Affecting on the Affinity of Receptor and the Phosphorylation of PP125^(FAK)

目的:为了进一步探讨血小板膜(GPⅡb)胞内段在信号传递中的作用。方法:选择表达GPⅡb^(R995A)Ⅲa的CHO细胞为研究对象,以正常人血小板和表达GPⅡbⅢa的CHO细胞为对照,应用流式细胞术、免疫沉淀、western blot-ting等方法检测PAC-1且结合能力和PP125^(FAK)磷酸化。结果:GPⅡbⅢa CHO细胞几乎不结合PAC-1,而GPⅡb^(R995A)ⅢaCHO细胞结合PAC-1明显增加;GPⅡbⅢa CHO细胞只有在粘附纤维蛋白原后才有FAK磷酸化,GP血b^(R995A)Ⅲa CHO细胞在悬浮时即有微弱的FAK磷酸化。结论:本研究提示GPⅡb^(R995A)点突变能改变受体的低亲合力状态,使受体具有高亲合性;该突变还介导细胞内信号传导,引起非配体结合的FAK磷酸化。

Objective: To investigate the roles of platelet membrane glycoprotein cytometry. Methods: Western blotting, Immunoprecipitation to detect the phosphorylation of PP125FAK and PAC-1 binding in Chinese hamster ovary (CHO) cell strains expressing wild-type GP Ⅱ b Ⅲ a/mutant GP Ⅱ bR995A Ⅲ a and normal platelets. Results: CHO cells bearing normal GP Ⅱ b Ⅲ a nearly failed to bind PAC-1, cells bearing mutant GP Ⅱ bK995A Ⅲa can bind PAC-1 ; and both phosphorylated PP125FAK when adherent to fibrinogen, but the mutant chimera exhibited the constitutive phosphorylation of PP125 when in suspension. Conclusion: The point mutation in GP Ⅱ bR995A can chang a low affinity state of the chimeric receptor, and demonstrate high affinity state; This mutation also mediate cytoplasmic signal transduction, induces phosphorylation of PP125FAK with no ligand binding.