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血管细胞的一般功能。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传导的理解，但重要的问题仍然存在。具体来说，哪些细胞内蛋白与整合素B细胞质尾部相互作用，它们是如何向整合素传递信号的？该项目的目标是使用先进的实验方法来测试与这些未解决问题相关的两个假设。第一种假设是，所有bps亲和力的由内而外调节是由talin和kindin -3到B3等蛋白质的协调募集控制的。这些蛋白之间的二元和三元相互作用将在活细胞（包括小鼠血小板）中进行检测，使用双分子荧光互补、FRET和原位接近连接。研究将探讨talin和kindin -3的募集在多大程度上依赖于Rap1 GTPase， kindin -3是否促进talin募集，反之亦然，以及粘附配体与所有bb3的结合是否足以促进这两种蛋白中的任何一种募集到B3尾部。第二种假设是，aV整合素B细胞质域与talin、kindlins和Src家族激酶（SFKs）的相互作用，无论是单独的还是联合的，都决定了aV介导的体内过程的结果。我们对斑马鱼胚胎进行了初步研究，使用morpholino寡核苷酸敲除aV，并使用aV mRNA来挽救敲除表型，结果表明，左右体轴规范所需的原肠胚形成事件依赖于aV，正如在aV敲除小鼠中报道的某些神经和血管发育事件一样。因此，将进行额外的敲除和拯救实验，以确定相关的斑马鱼整合素aV B亚基调节侧性规范。为了确定整合素是否与talin、kindlins或sfk相互作用，将对突变p亚基进行拯救实验，这些突变p亚基被预测并证明可以选择性或集体破坏与这些蛋白质的相互作用。拟议的研究应阐明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，对人类血小板和血管生物学具有重要意义。

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.