权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Characterization of regulatory proteins for β3 integrins.

β3 整合素调节蛋白的表征。

基本信息

批准号：
15390299
负责人：
TOMIYAMA Yoshiaki
金额：
$ 8.77万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15390299/
关键词：
integrin CMK inside-out signaling ligand-binding sites PKC WAVE differential display法 cDNAマイクロアレイ

项目摘要

β3 integrins, which contain αIIbβ3 and αvβ3, play critical roles in atherosclerosis as well as thrombosis.Therefore, the elucidation of the regulatory mechanisms for integrin function is a critical issue to control these vascular events. In this research project, we have tried to establish a new experimental model other than platelets to examine the regulation of β3 integrin function and their ligand-binding sites.We examined ligand-binding sites within the αvβ-propeller domain employing alanine-scanning mutagenesis within W3 and W4 loops. We demonstrated that 178Tyr and Asp128 are the critical residues for ligand-binding.. In addition Ala215Tyr αv increased the integrin affinity.We next demonstrated that in a megakaryocytic cell line, CMK,CD42b(GPIb)-positive cells could activate αIIbβ3 after PMA stimulation. We then searched molecules responsible for integrin activation employing differential display method and/or cDNA micrarray During CMK maturation expression levels of 306 genes increased. Among them we were interested in β1-tublin, CKIP-1, cortactin, PKCδ, PKCβ1, WAVE-1, Rab27B etc. Employing bryostatin-1 we suggested that PKCα and β play a role in integrin activation. Employing primary megakaryocytic cells derived from cord blood, we confirmed that WAVE-1 expression, but not WAVE-2 or -3, increased during megakaryocytic differentiation. In adhered platelets both WAVE-1 and WAVE-2 were localized at the edge of the lamellipodia, suggesting that these molecules may regulate actin reorganization during platelet spreading.

β3整合素（包括αIIbβ3和αvβ3）在动脉粥样硬化和血栓形成中起重要作用，因此，阐明整合素的功能调控机制是控制这些血管事件的关键。本研究尝试建立一种不同于血小板的新的实验模型来研究β3整合素的功能调控及其配体结合位点，采用丙氨酸扫描突变技术对αvβ-螺旋桨结构域W3和W 4环的配体结合位点进行了研究。我们证明了178 Tyr和Asp 128是配体结合的关键残基。此外，Ala 215 Tyr αv增加了整合素的亲和力。我们接下来证明，在巨核细胞系中，CMK，CD 42 b（GPIb）阳性细胞可以在PMA刺激后激活αIIbβ3。然后，我们使用差异显示方法和/或cDNA微阵列寻找负责整合素激活的分子。在CMK成熟过程中，306个基因的表达水平增加。其中，β1-tublin、CKIP-1、coronin、PKCδ、PKCβ1、WAVE-1、Rab 27 B等是我们感兴趣的研究对象。采用来自脐带血的原代巨核细胞，我们证实了WAVE-1的表达，而不是WAVE-2或-3，在巨核细胞分化过程中增加。在粘附的血小板中，WAVE-1和WAVE-2都位于板状伪足的边缘，这表明这些分子可能在血小板铺展过程中调节肌动蛋白重组。