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，双向信号似乎是大多数细胞的普遍功能。虽然对B3整合素allbB3和aVB3的研究有助于我们目前对整合素信号的理解，但重要的问题仍然存在。具体地说，哪些细胞内蛋白与整合素B胞浆尾巴相互作用，以及它们如何向整合素传递信号以及如何从整合素传递信号？这个项目的目标是使用先进的实验方法来测试与这些悬而未决的问题相关的两个假设。第一个假说是，ALLBP亲和力的由内向外调节是由Talin和kindlin-3等蛋白质协调招募到B3控制的。这些蛋白质之间的二元和三元相互作用将在活细胞中进行检验，包括小鼠血小板，使用双分子荧光互补、FRET和原位邻近连接。研究将解决talin和kindlin-3招募对Rap1 GTP酶的依赖程度，kindlin-3是否促进talin招募，反之亦然，以及与allbB3结合的粘性配体是否足以促进这两种蛋白质中的任何一种向B3尾部招募。第二个假设是，AV整合素B胞浆结构域与talin、kindlins和Src家族激酶(SFK)的相互作用，无论是单独还是联合，都决定了AV介导的体内过程的结果。我们对斑马鱼胚胎的初步研究表明，左右体轴指定所需的原肠形成事件与AV有关，在AV基因敲除小鼠中也报道了某些神经和血管发育事件。因此，将进行额外的击倒和挽救实验，以确定相关的斑马鱼整合素AV B亚单位，以调节偏侧性的规范。为了确定是否涉及整合素与talin、kindlins或sfk的相互作用，将用突变的p亚基进行救援实验，这些突变的p亚基被预测和证明选择性地或集体地干扰与这些蛋白质的相互作用。建议的研究将阐明Allb和AV整合素信号的基本和保守机制，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.