Calpain and beta3 cytoplasmic domain in platelet integrin signaling

血小板整合素信号传导中的钙蛋白酶和 β3 胞质结构域

• 批准号: 7213806
• 负责人: Xiaoping Du
• 金额: $ 34.93万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-12-15 至 2010-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7213806
• 关键词: Adhesions; Binding; Blood Platelets; C-terminal; Calcium; Calpain; Calpain I; Calpain II; Cell Adhesion; Cell model; Cell-Cell Adhesion; Cells; Cleaved cell; Cytoplasmic Tail; Cytoskeleton; Endopeptidases; Family; Glycoprotein Ib; Human; In Vitro; Integrin beta3; Integrins; Knock-in Mouse; Knockout Mice; Ligand Binding; Mammalian Cell; Mediating; Modeling; Mus; Mutagenesis; Peptide Hydrolases; Phosphorylation; Physiological; Platelet Activation; Play; Proteins; Recombinants; Regulation; Resistance; Role; Signal Transduction; Site; Small Interfering RNA; Structure; Talin; Techniques; Testing; Tyrosine Phosphorylation; adhesion receptor; base; mutant; reconstitution; response

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.

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发挥关键作用。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由内而外的信号传导，激活 allbbeta3 的配体结合功能。配体与 allbbeta3 结合会诱导由外向内的信号传导，这也需要整合素胞质结构域来介导细胞反应，例如蛋白质酪氨酸磷酸化、细胞骨架重组、细胞扩散和稳定粘附。我们在表达重组人整合素IIbbeta3和人糖蛋白Ib-IX的培养哺乳动物细胞模型中重建了β3整合素信号传导。使用这个模型，我们提供了证据，证明由外向内信号传递的结构要求与由内向外信号传递不同。因此，我们假设β3整联蛋白的由外向内信号传导和由内向外信号传导可以受到差异性调节。此外，我们还发现，整合素的胞质结构域可以被钙依赖性蛋白酶钙蛋白酶在两个功能重要的 NXXY 基序侧翼的特定位点裂解，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差异调节由内向外和由外向内信号传导的重要机制。特别是，Y759 处的钙蛋白酶裂解选择性地调节由外向内的信号传导。为了检验这些假设，我们建议研究（1）为什么从内到外和从外到内的整合素信号传导需要β3细胞质结构域中的共同和不同的结构，（2）钙蛋白酶识别β3的结构基础，（3）钙蛋白酶裂解是否受酪氨酸磷酸化调节，以及（4）使用钙蛋白酶I敲除小鼠和钙蛋白酶I敲除小鼠和钙蛋白酶是否以及如何调节整合素信号传导。 siRNA 技术和钙蛋白酶抗性突变体整合素。