Calpain and beta3 cytoplasmic domain in platelet integrin signaling

描述(申请人提供)：一种血小板整合素，allbbeta3，在血小板黏附和聚集中起关键作用。整合素allbbeta3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内向外信号，激活allbbeta3的配体结合功能。与allbbeta3的配体结合可诱导由外向内的信号转导，这也需要整合素的胞浆结构域来介导细胞反应，如蛋白酪氨酸磷酸化、细胞骨架重组、细胞扩散和稳定的黏附。我们已经在培养的哺乳动物细胞模型中重组了表达重组人整合素11bbeta3和人糖蛋白Ib-IX的整合素信号。使用这个模型，我们提供了证据，证明了自外向内信令的结构要求不同于自内向外信令。因此，我们假设Beta3整合素的自外向内信号和自内向外信号可以受到不同的调节。此外，我们还发现，整合素的细胞质结构域可以被钙依赖的蛋白酶calain在两个功能重要的NXXY基序两侧的特定位置切割，从而导致整合素双向信号的差异调节。因此，我们进一步假设，Calain裂解作为一种重要的机制，对由内而外和由外而内的信号进行了不同的调节。特别是，Y759处的Calain裂解选择性地调节由外向内的信号。为了检验这些假说，我们建议研究(1)为什么Beta3的细胞质区域中自内向外和由外向内的整合素信号需要共同和不同的结构，(2)Beta3识别calain的结构基础，(3)calain的切割是否受酪氨酸磷酸化的调控，以及(4)使用calain I基因敲除小鼠和siRNA技术以及calain抗性突变体整合素是否以及如何调控整合素信号。

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.