New Approaches to Interrogate Platelet and Vascular Integrins

血管细胞，包括血小板，表达大量的整合素，和双向信号似乎是他们中的大多数的一般功能。虽然对B3整合素allbB 3和aVB 3的研究有助于我们目前对整合素信号传导的理解，但仍然存在重要的问题。具体而言，哪些细胞内蛋白质与整合素B胞质尾相互作用，它们如何将信号传递给整合素和从整合素传递信号？本项目的目标是使用先进的实验方法来测试与这些未解决的问题相关的两个假设。第一个假设是allbps亲和力的由内而外调节由talin和kindlin-3等蛋白质向B3的协调募集控制。这些蛋白质之间的二元和三元相互作用将在活细胞，包括小鼠血小板，使用双分子荧光互补，FRET，和原位邻近连接检查。研究将解决talin和kindlin-3募集依赖于Rap 1 GTdR的程度，kindlin-3是否促进talin募集或反之亦然，以及与allbB 3结合的粘附配体是否足以促进这些蛋白质中的任一种募集到B3尾。第二个假设是，α V整联蛋白B胞质结构域与talin、kindlin和Src家族激酶（SFK）的相互作用（单独或组合）决定了α V介导的体内过程的结果。我们的初步研究与斑马鱼胚胎使用吗啉代寡核苷酸敲低aV，和aV mRNA拯救敲低表型，揭示了原肠胚事件所需的左右体轴规格依赖于aV，因为某些神经和血管发育事件也报道在aV敲除小鼠。因此，将进行额外的敲低和拯救实验以鉴定调节偏侧性特化的相关斑马鱼整联蛋白aV B亚基。为了确定是否涉及整联蛋白与talin、kindlin或SFK的相互作用，将用预测并证明选择性或共同破坏与这些蛋白质的相互作用的突变体p亚基进行拯救实验。拟议的研究应该澄清allb和aV整合素信号传导的基本和保守机制，并为基因靶向小鼠的后续研究提供信息，并对人类血小板和血管生物学产生影响。

Vascular cells, including platelets, express numerous integrins, and bidirectional signaling appears to be a general function of most of them. While studies of B3 integrins allbB3 and aVB3 have contributed to our current understanding of integrin signaling, important questions remain. Specifically, which intracellular proteins interact with integrin B cytoplasmic tails and how do they transmit signals to and from integrins? The goal of this project is to test two hypotheses relevant to these unresolved questions using advanced experimental approaches. The first hypothesis is that inside-out regulation of allbps affinity is controlled by the coordinated recruitment of proteins such as talin and kindlin-3 to B3. Binary and ternary interactions among these proteins will be examined in living cells, including murine platelets, using bimolecular fluorescence complementation, FRET, and in situ proximity ligation. Studies will address the degree to which talin and kindlin-3 recruitment are dependent on Rap1 GTPase, whether kindlin-3 promotes talin recruitment or vice-versa, and whether adhesive ligand binding to allbB3 is sufficient to promote recruitment of either of these proteins to the B3 tail. The second hypothesis is that interactions of the aV integrin B cytoplasmic domain with talin, kindlins and Src family kinases (SFKs), either alone or in combination, dictate the outcome of aV-mediated processes in vivo. Our preliminary studies with zebrafish embryos using morpholino oligonucleotides to knockdown aV, and aV mRNA to rescue knockdown phenotypes, reveal that gastrulation events required for left-right body axis specification are dependent on aV, as are certain neurological and vascular developmental events also reported in aV knockout mice. Therefore, additional knockdown and rescue experiments will be carried out to identify the relevant zebrafish integrin aV B subunit that regulates specification of laterality. To determine whether integrin interactions with talin, kindlins or SFKs are involved, rescue experiments will be conducted with mutant p subunits that are predicted and demonstrated to selectively or collectively disrupt interactions with these proteins. The proposed studies should clarify basic and conserved mechanisms of allb and aV integrin signaling and inform followup studies in gene-targeted mice, with implications for human platelet and vascular biology.