Regulation of Outside-in Integrin Signaling in Platelets

血管损伤后，黏附配体如纤维蛋白原和血管性血友病因子结合整合素 Allb/β3在止血和血栓形成过程中影响血小板聚集和扩散。这些回应是 由配体介导的Allb/？3簇触发，启动由外向内的信号来重组肌动蛋白 细胞骨架。该项目的最新研究已经确定，血小板中由外向内的信号需要Src 家族酪氨酸激酶(SFK)，特别是c-Src，与/？3结合并被Allb/？3簇激活。 依赖于PTP-1B的方式，PTP-1B是一种蛋白质酪氨酸磷酸酶。在这里，三个主要的悬而未决的问题 将被问及血小板中由外向内信号的分子基础及其生物学 后果。首先，整合素和SFK之间的直接相互作用是否代表了一种通用的机制 血小板内自外向内信号的时空启动？由于血小板含有五种不同的整合素 和至少六种不同的SFK，这种可能性将通过免疫共沉淀技术进行评估，通过 活细胞内双分子荧光互补成像及活体内Src激活的定位 细胞使用基于FRET的记者。此外，还将评估果蝇细胞中整合素/SFK的相互作用 确定整合素直接激活SFK在多大程度上是一个进化保守的过程。 第二，PTP-1B如何激活整合素下游的c-Src？PTP-1B的作用机制 被招募到Allb/β3/c-Src复合体，以及可能被招募到其他整合素/SFK复合体，将在#年进行评估 血小板和模型细胞系统，重点研究适配蛋白的可能作用。此外，这一影响， 将测定整合素聚集在PTP-1B上的催化活性。第三，是否有选择地颠覆外部 信号影响体内血栓的形成？在这里，动脉血栓的研究将在新的基因靶点上进行 预测c/LLB/83与c-Src或其他SFK相互作用有选择性缺陷的小鼠，或 肌动蛋白重组所需的下游事件。总之，这些研究将提供分子洞察力。 了解外-内整合素信号是如何启动的，并确定这一过程的调节程度 体内的血小板功能。因此，这一研究路线可能会导致新的抗血栓药物的鉴定 并作为其他血细胞中整合素信号传递的范例。

Following vascular injury, adhesive ligands such as fibrinogen and von Willebrand factor engage integrin allb/?3 to effect platelet aggregation and spreading during hemostasis and thrombosis. These responses are triggered by ligand-mediated allb/?3 clustering, which initiates "outside-in" signals to reorganize the actin cytoskeleton. Recent work from this project has established that outside-in signaling in platelets requires Src family tyrosine kinases (SFKs), c-Src in particular, which bind to /?3 and are activated by allb/?3 clustering in a manner dependent on PTP-1B, a protein tyrosine phosphatase. Here, three major unresolved questions will be asked concerning the molecular basis of outside-in signaling in platelets and its biological consequences. First, do direct interactions between integrins and SFKs represent a general mechanism for spatio-temporal initiation of outside-in signaling in platelets? Since platelets contain five different integrins and at least six different SFKs, this possibility will be evaluated by co-immunoprecipitation techniques, by bimolecular fluorescence complementation imaging in live cells, and by localization of Src activation in live cells using a FRET-based reporter. In addition, integrin/SFK interactions will be assessed in Drosophila cells to determine the extent to which direct activation of SFKs by integrins is an evolutionarily conserved process. Second, how does PTP-1B activate c-Src downstream of integrins? The mechanism by which PTP-1B is recruited to the allb/?3/c-Src complex, and possibly to other integrin/SFK complexes, will be evaluated in platelets and model cell systems, focusing on the possible role of adapter proteins. In addition, the effect of integrin clustering on PTP-1B catalytic activity will be determined. Third, does selective disruption of outsidein signaling affect thrombus formation in vivo? Here arterial thrombosis will be studied in novel gene-targeted mice predicted to have selective defects in the interaction of c/llb/83 with c-Src or other SFKs, or defects in downstream events required for actin reorganization. Altogether, these studies will provide molecular insights into how outside-in integrin signaling is initiated and establish the extent to which this process regulates platelet function in vivo. Thus, this line of investigation may lead to identification of new anti-thrombotic drug targets and serve as a paradigm for integrin signaling in other blood cells.