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Analysis of GBP receptor structure and GBP signal transduction in hemocytes

血细胞GBP受体结构及GBP信号转导分析

基本信息

批准号：
18370031
负责人：
HAYAKAWA Yoichi
金额：
$ 11.56万
依托单位：
Saga University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2009
项目状态：
已结题

项目摘要

Growth-blocking peptide (GBP) is an insect cytokine that stimulates a class of immune cells called plasmatocytes to adhere to each other and a foreign surface. Although structure-activity studies have been extensively performed in GBP and its mutants of Lepidoptera Pseudaletia separata, the signaling pathway of GBP-dependent activation of plasmatocytes remains unknown. We identified a novel adaptor protein (P77) with a molecular mass of 77kDa containing SH2/SH3 domain binding motifs and an immunoreceptor tyrosine-based activation motif (ITAM)-like domain in the cytoplasmic region of the C-terminus. Although P77 did not have the capacity for direct binding with GBP, its cytoplasmic tyrosine residues were specifically phosphorylated within seconds after addition of GBP to a plasmatocyte suspension. Tyrosine phosphorylation of P77 was also observed when hemocytes were incubated with Enterobactor cloacae or Micrococcus luteus, but this phosphorylation was found to be induced by GBP released from hemocytes stimulated by the pathogens. We further detected tyrosine phosphorylation of the integrin β subunit in plasmatocytes stimulated by GBP. Double-stranded RNAs targeting P77 not only decreased GBP-dependent tyrosine phosphorylation of the integrin β subunit, but also abolished the GBP-induced spreading of plasmatocytes on foreign surfaces. P77 RNAi larvae also showed significantly higher mortality than control larvae following infection with Serratia marcescens, thus indicating that P77 is essential for GBP to mediate a normal innate cellular immunity in insects. These results demonstrated that GBP signaling in plasmatocytes requires the adaptor protein P77 and that active P77-assisted tyrosine phosphorylation of integrins is critical for the activation of plasmatocytes.

生长阻断肽（GBP）是一种昆虫细胞因子，可刺激一类称为浆细胞的免疫细胞相互粘附并粘附在异物表面。尽管对鳞翅目昆虫Pseudaletia separata的GBP及其突变体进行了广泛的构效关系研究，但GBP依赖的浆细胞活化信号通路仍不清楚。我们鉴定了一种分子量为77 kDa的新型衔接蛋白（P77），其C-末端的胞质区域含有SH 2/SH 3结构域结合基序和基于免疫受体酪氨酸的激活基序（ITAM）样结构域。虽然P77没有直接与GBP结合的能力，但其胞质酪氨酸残基在将GBP加入浆细胞悬液后数秒内特异性磷酸化。当血细胞与阴沟肠杆菌或藤黄微球菌孵育时，也观察到P77的酪氨酸磷酸化，但发现这种磷酸化是由病原体刺激血细胞释放的GBP诱导的。我们进一步检测了GBP刺激的浆细胞中整合素β亚基的酪氨酸磷酸化。靶向P77的双链RNA不仅降低了整合素β亚基依赖于GBP的酪氨酸磷酸化，而且消除了GBP诱导的浆细胞在外源表面上的扩散。感染粘质沙雷氏菌后，P77 RNAi幼虫的死亡率也显著高于对照幼虫，这表明P77对于GBP介导昆虫正常的先天细胞免疫是必需的。这些结果表明，浆细胞中的GBP信号传导需要衔接蛋白P77，并且整合素的活性P77辅助的酪氨酸磷酸化对于浆细胞的激活至关重要。