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| 甲状旁腺素通过磷脂酶C非依赖途径激活蛋白激酶C |
| Parathyroid hormone activates protein kinase C via phospholipase C-independent signaling pathway |
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| DOI： |
| 中文关键词: 甲状旁腺素 荧光共振能量转移 PKC激活报告分子 PKC-delta激活报告分子 甲状旁腺素I型受体 |
| 英文关键词:Parathyroid hormone Fluorescence resonance energy transfer C kinase activity reporter C-delta kinase activity reporter Type I PTH receptor |
| 基金项目:广东省自然科学基金项目(9151051501000087)，国家自然科学基金(30973061) |
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| 中文摘要: |
| 目的 利用基于荧光共振能量转移(Fluorescence resonance energy transfer，FRET)技术的检测PKC激活或PKC-delta激活的报告分子来确定PTH是否可以通过PLC非依赖途径激活PKC和PKC-delta。方法 将表达PKC激活报告分子(CKAR)的质粒和表达PKG-delta激活报告分子的质粒转染HEK293细胞，培养72h后通过共聚焦显微镜检测FRET的改变，并以此判断佛波酯(TPA)是否激活PKC和PKC-delta。将表达甲状旁腺素l型受体(PTHRl)的质粒与CKAR质粒或PKC-delta质粒共转染HEK293细胞，培养72h后通过共聚焦显微镜检测FRET的改变。并判断PTH(1-34)、GIRl9(1-34)和0.1％的三氟乙酸(TFA)对PKC和PKC-delta的作用。结果 在转染CKAR质粒或PKC-delta质粒的HEK293细胞，TPA均使青色荧光与黄色荧光的强度之比(C/Y)增加。在共转染PTH1R质粒与CKAR质粒或PKC-delta质粒的HEK293细胞，PTH(1-34)和G1R19(104)均增加了C/Y的值，而0.1％TFA未引起C/Y的改变。结论PTH与PTHR1结合后通过PLC非依赖途径激活PKC/PKC-delta。 |
| 英文摘要: |
| Objective To determine whether PTH activates PKC／PKC-delta via PLC-independent pathway using the reporter of PKC or PKC-delta activation based on fluorescence resonance energy transfer(FRET)．Methods HEK293 cells were transfected with C kinase activity reporter(CKAR) or C-delta kinase activity reporter(PKC-delta)．After incubation for 72h，FRET
was measured with or without stimulation of phorbol esters(TPA)．The activation of PKC or PKC-delta could be judged by the change in FRET．HEK293 cells were co-transfected with either CKAR or PKC-delta plasmid or PTHRI plasmid．Stimulation of PKC or PKC-delta by PTH(1-34) or G1R19(1-34)or0.1％ trifluoroacetic acid(TFA) were demonstrated by monitoring the change in FRET after 72 h．Results TPA increased the CFP/YFP emission ratio(C/Y) in HEK293 cells transfected with CKAR or PKC-delta plasmid．PTH(1-34) and GIRl9(1-34), bat not 0.1％TFA，increased C/Y in HEK293 cells co-transfected with either CKAR or PKC-delta plasmid or PTHRl plasmid．Conclusion PTH binding to PTHRl can activate PKC and PKC-delta via PLC-independent signaling pathway． |
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