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。第一个假设是，所有bps亲和力的由内而外调节是由蛋白质的协调募集控制的

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 allbp3 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.