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目的:检测人类血栓素α受体(TPα)C端丝氨酸/苏氨酸残基的各种两氨酸诱变体作为PKC底物的能力,以确定被PKC磷酸化和脱敏的特异的丝氨酸/苏氨酸残基.方法:为了易于鉴定被磷酰化的细胞内区段,使用甘胱甘肽S-转移酶(GST)-细胞内区段融合蛋白作纯化的PKC底物,然后突变磷酰化蛋白的cDNA,以找出TPα被PKC磷酰化的主要部位,并将有组氨酸尾野生型或诱变型血栓素受体α稳定地转移到人类胚胎肾(HEK)293细胞,以研究该受体的磷酸化和脱敏.结果:仅C-端能充当纯化PKC底物.丝氨酸-331(mP4)被显示强的磷酰化,丝氨酸-324(mP1)则轻微磷酰化,丝氨酸-329(mP3)则微弱磷酰化,而其它丝氨酸/苏氨酸残基没被发现磷酰化.佛被醇-12-肉豆蔻酸酯-13-乙酸盐(PMA)诱导表达野生型TPα的HEK 293细胞磷酰化.然而不能显示触发表达丝氨酸331丙氨酸诱变受体的HEK 293受体磷酸化.用PMA预处理表达野生型受体的HEK 293细胞能抑制I-B0P诱导Ca~(2+)释放,然而用PMA预处理表达诱变受体的细胞不能中止I-BOP抑制Ca~(2+)释放.结论:丝氨酸331是TPαa磷酰化和脱敏的主要和关键部位.
PURPOSE: To examine the ability of various glutamic acid mutants on the C-terminal serine / threonine residues of human thromboxane A receptor (TPα) as PKC substrates to identify specific serines phosphorylated and desensitized by PKC / Threonine residues.Methods: To facilitate the identification of phosphorylated intracellular segments, a purified PKC substrate was prepared using a glutathione S-transferase (GST) - intracellular segment fusion protein and then mutated Phosphorylated protein to find the major site of TPα phosphorylation by PKC and stably transfer the histidine tail wild-type or mutagenized thromboxane receptor α to human embryonic kidney (HEK) 293 cells , To study phosphorylation and desensitization of this receptor.Results: Only the C-terminus could act as a purified PKC substrate.Salon-331 (mP4) was shown to be strongly phosphorylated while serine-324 (mP1) was slightly phosphorylated , While mP3 was weakly phosphorylated while no other serine / threonine residues were found to be phosphorylated.Fol was induced by alcohol-12-myristate-13-acetate (PMA) Phosphorylation of HEK 293 cells expressing wild-type TP [alpha], however, did not appear to trigger HEK 293 receptor phosphorylation that triggers the expression of alanine-331 alanine mutagenized receptor The pretreatment of HE expressing 293 wild-type receptor with PMA K 293 cells could inhibit I-B0P-induced Ca 2+ release, however, pretreatment of PMA-expressing cells with PMA did not stop the I-BOP inhibition of Ca 2+ release.Conclusion: Serine 331 is TPαa phosphorus Acylation and desensitization of the main and key parts.