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起关键作用。整合素AllbBeta3介导双向信号传导转导。细胞内信号通过整合素细胞质结构域诱导内外信号传导，从而激活AllBBETA3的配体结合函数。配体与AllBBETA3结合诱导外部信号传导，这也需要整联蛋白的细胞质结构域介导细胞反应，例如蛋白酪氨酸磷酸化，细胞骨架重组，细胞扩散，细胞扩散和稳定的粘附。我们已经在表达重组人整合素LLBBETA3和人糖蛋白IB-IX的培养的哺乳动物细胞模型中重新组成了β3整合素信号传导。使用此模型，我们提供了证据，表明外部信号传导的结构要求与内部信号传导不同。因此，我们假设可以差异地调节β3整合素的外部信号传导和内部信号传导。此外，我们已经表明，整联蛋白的细胞质结构域可以通过钙依赖性蛋白酶钙蛋白酶裂解，在两个功能上重要的NXXY基序的特定位置，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，可以差异地调节内外和外部信号传导。特别是，Y759处的钙蛋白酶切割有选择地调节外部信号传导。要检验这些假设，我们建议研究（1）为什么在内外和外内的整合素信号传导和外部整合蛋白信号传导中需要β3的细胞质结构域中的常见和不同结构，（2）钙蛋白酶识别β3的结构基础，（3）（3）Calpain裂解是否通过硫氨酸磷酸化和（4）的cal量（4）callation intranpiant和4）调节（3） siRNA技术和耐钙蛋白酶的突变蛋白。