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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，可以刺激一类叫做浆细胞的免疫细胞相互粘附并粘附在异物表面。尽管对假分离鳞翅目GBP及其突变体进行了广泛的结构-活性研究，但GBP依赖性浆细胞激活的信号通路尚不清楚。我们鉴定了一种分子质量为77kDa的新型接蛋白（P77），它含有SH2/SH3结构域结合基序和位于c端细胞质区域的基于免疫受体酪氨酸的激活基序（ITAM）样结构域。虽然P77不具有与GBP直接结合的能力，但在将GBP添加到浆细胞悬液中后，其细胞质酪氨酸残基在几秒钟内被特异性磷酸化。在与阴沟肠杆菌或黄体微球菌孵育血细胞时，也观察到P77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gbp依赖性的整合素β亚基酪氨酸磷酸化，而且还消除了gbp诱导的浆细胞在外源表面的扩散。在粘质沙雷菌感染后，P77 RNAi幼虫的死亡率也明显高于对照幼虫，这表明P77对于GBP介导昆虫正常的先天细胞免疫至关重要。这些结果表明，血浆细胞中的GBP信号需要适配蛋白P77，而P77辅助的酪氨酸磷酸化对于血浆细胞的激活至关重要。