Lysophosphatidylcholines activate G2A inducing G(αi)₋₁-/G(αq/)₁₁- Ca²(+) flux, G(βγ)-Hck activation and clathrin/β-arrestin-1/GRK6 recruitment in PMNs.
Lysophosphatidylcholines activate G2A inducing G(αi)₋₁-/G(αq/)₁₁- Ca²(+) flux, G(βγ)-Hck activation and clathrin/β-arrestin-1/GRK6 recruitment in PMNs.
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DOI：
10.1042/bj20091087
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发表时间：
2010-11-15
期刊：
影响因子：
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通讯作者：
Silliman CC
中科院分区：
文献类型：
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作者：
Khan SY;McLaughlin NJ;Kelher MR;Eckels P;Gamboni-Robertson F;Banerjee A;Silliman CC
Lyso-PC（溶血磷脂酰胆碱）是一种脂质混合物，在细胞血液成分储存过程中积累，与 TRALI（输血相关急性肺损伤）有关，并直接影响中性粒细胞 [PMN（多形核白细胞）] 的生理机能。由于 PMN 上表达的 G2A 受体据报道可识别 lyso-PC，因此我们推测 G2A 的 lyso-PC 激活通过释放 Gα 和 Gβγ 亚基、激酶激活以及将网格蛋白、β-arrestin-1 和 GRK6（G 蛋白受体激酶 6）募集至 G2A 来进行信号转导，从而导致胞质 Ca2+ 增加。通过标准技术分离 PMN，用 lyso-PC 引发 5-180 秒，然后裂解用于蛋白质印迹分析、免疫沉淀或亚细胞分级分离，或固定并涂在载玻片上用于数字显微镜检查。结果表明，lyso-PC 通过 S-磷酸化和内化引起 G2A 受体快速激活，导致 Gαi-1 和 Gαq/11 释放，从而导致胞质 Ca2+ 增加，而 G2A 抗体或这些亚基的细胞内中和可抑制胞质 Ca2+ 的增加。 Lyso-PC 还引起 Gβγ 亚基的释放，该亚基表现出与激活的 Hck（造血细胞激酶；Tyr411）的物理相互作用 (FRET+)。此外，G2A招募了网格蛋白、β-arrestin-1和GRK6：网格蛋白对于信号转导很重要，GRK6对于受体脱敏，而β-arrestin-1既传播又终止信号。我们得出结论，G2A 的 lyso-PC 激活引起 Gαi-1、Gαq/11 和 Gβγ 的释放，导致胞质 Ca2+ 流动、Hck 激活以及网格蛋白、β-arrestin-1 和 GRK6 的募集。
Lyso-PCs (lysophosphatidylcholines) are a mixture of lipids that accumulate during storage of cellular blood components, have been implicated in TRALI (transfusion-related acute lung injury) and directly affect the physiology of neutrophils [PMNs (polymorphonuclear leucocytes)]. Because the G2A receptor, expressed on PMNs, has been reported to recognize lyso-PCs, we hypothesize that lyso-PC activation of G2A causes the increases in cytosolic Ca2+ via release of Gα and Gβγ subunits, kinase activation, and the recruitment of clathrin, β-arrestin-1 and GRK6 (G-protein receptor kinase 6) to G2A for signal transduction. PMNs were isolated by standard techniques, primed with lyso-PCs for 5–180 s, and lysed for Western blot analysis, immunoprecipitation or subcellular fractionation, or fixed and smeared on to slides for digital microscopy. The results demonstrated that lyso-PCs cause rapid activation of the G2A receptor through S-phosphorylation and internalization resulting in Gαi-1 and Gαq/11 release leading to increases in cytosolic Ca2+, which was inhibited by an antibody to G2A or intracellular neutralization of these subunits. Lyso-PCs also caused the release of the Gβγ subunit which demonstrated a physical interaction (FRET+) with activated Hck (haemopoietic cell kinase; Tyr411). Moreover, G2A recruited clathrin, β-arrestin-1 and GRK6: clathrin is important for signal transduction, GRK6 for receptor de-sensitization, and β-arrestin-1 both propagates and terminates signals. We conclude that lyso-PC activation of G2A caused release of Gαi-1, Gαq/11 and Gβγ, resulting in cytosolic Ca2+ flux, Hck activation, and recruitment of clathrin, β-arrestin-1 and GRK6.