Calpain and beta3 cytoplasmic domain in platelet integrin signaling

描述（由申请人提供）：血小板整合素allbbeta3在血小板粘附和聚集中起关键作用。整合素介导双向信号转导。胞内信号通过整合素胞质结构域诱导内外信号，激活allbbeta3的配体结合功能。配体与allbbeta3结合诱导外向内信号传导，这也需要整合素细胞质结构域介导蛋白酪氨酸磷酸化、细胞骨架重组、细胞扩散和稳定粘附等细胞反应。我们在表达重组人整合素lbbeta3和人糖蛋白Ib-IX的培养哺乳动物细胞模型中重组了β a3整合素信号通路。使用这个模型，我们已经提供了证据，表明由外向内信号的结构要求不同于由内向外信号。因此，我们假设β 3整合素的由外向内信号和由内向外信号可能受到不同的调节。此外，我们已经证明，整合素的细胞质结构域可以被钙依赖性蛋白酶calpain在两个功能重要的NXXY基序两侧的特定位点切割，从而导致整合素双向信号传导的差异调节。因此，我们进一步假设钙蛋白酶裂解是一种重要的机制，可以调节由内向外和由外向内信号传导的差异。特别是，Y759位点的calpain切割选择性地调控了外向内信号传导。为了验证这些假设，我们建议研究(1)为什么β 3胞质区域的共同和独特结构需要由内向外和由外向内整合素信号传导；(2)β 3的calpain识别的结构基础；(3)calpain的裂解是否受酪氨酸磷酸化的调节；(4)calpain是否以及如何通过calpain I敲除小鼠和siRNA技术以及calpain抗性整合素突变体来调节整合素信号传导。

DESCRIPTION (provided by applicant): A platelet integrin, allbbeta3, plays critical roles in platelet adhesion and aggregation. Integrin allbbeta3 mediates two-way signaling transduction. Intracellular signals, via the integrin cytoplasmic domains induces inside-out signaling, activating the ligand binding function of allbbeta3. Ligand binding to allbbeta3 induces outside-in signaling, which also requires integrin cytoplasmic domains to mediate cellular responses such as protein tyrosine phosphorylation, cytoskeleton reorganization, cell spreading, and stable adhesion. We have reconstituted beta3 integrin signaling in cultured mammalian cell model expressing recombinant human integrin llbbeta3 and human glycoprotein Ib-IX. Using this model, we have provided evidence that the structural requirements for outside-in signaling are different from inside-out signaling. Thus we hypothesize that outside-in signaling and inside-out signaling of beta3 integrins can be differentially regulated. Furthermore, we have shown that the cytoplasmic domain of integrin can be cleaved by the calcium-dependent protease, calpain, at specific sites flanking two functionally important NXXY motifs, causing differential regulation of integrin two-way signaling. Thus we further hypothesize that calpain cleavages serve as an important mechanism that differentially regulates inside-out and outside-in signaling. In particular, calpain cleavage at Y759 selectively regulates outside-in signaling. To test these hypotheses, we propose to investigate (1) why common and distinct structures in the cytoplasmic domain of beta3 are required in inside-out and outside-in integrin signaling, (2) the structural basis of calpain recognition of beta3, (3) whether calpain cleavage is regulated by tyrosine phosphorylation, and (4) whether and how calpain regulate integrin signaling using calpain I knockout mice and siRNA techniques and a calpain-resistant mutant the integrin.