Calpain and beta3 cytoplasmic domain in platelet integrin signaling

描述（由申请人提供）：血小板整合素allb β 3在血小板粘附和聚集中起关键作用。整合素allb β 3介导双向信号转导。细胞内信号通过整合素胞质结构域诱导由内向外的信号传导，激活allb β 3的配体结合功能。与allb β 3结合的配体诱导由外向内信号传导，这也需要整联蛋白胞质结构域来介导细胞应答，如蛋白酪氨酸磷酸化、细胞骨架重组、细胞铺展和稳定粘附。我们已经在表达重组人整联蛋白IIb β 3和人糖蛋白Ib-IX的培养的哺乳动物细胞模型中重建了β 3整联蛋白信号传导。使用这个模型，我们已经提供了证据表明，结构要求由外向内的信号是不同的从内向外的信号。因此，我们假设β 3整合素的由外向内信号传导和由内向外信号传导可以被差异调节。此外，我们已经表明，整合素的胞质结构域可以被钙依赖性蛋白酶，钙蛋白酶，在特定的网站侧翼的两个功能重要的NXXY图案，导致差异调节整合素的双向信号传导。因此，我们进一步假设，钙蛋白酶裂解作为一个重要的机制，差异调节由内而外和由外而内的信号。特别地，在Y 759处的钙蛋白酶切割选择性地调节由外向内信号传导。为了验证这些假设，我们建议研究（1）为什么β 3胞质结构域中的共同和不同结构在由内向外和由外向内的整合素信号传导中是必需的，（2）钙蛋白酶识别β 3的结构基础，（3）钙蛋白酶切割是否受酪氨酸磷酸化调节，以及（4）使用钙蛋白酶I敲除小鼠和siRNA技术以及钙蛋白酶抗性突变体整联蛋白是否以及如何调节整联蛋白信号传导。

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.