New Approaches to Interrogate Platelet and Vascular Integrins

检测血小板和血管整合素的新方法

• 批准号: 8256549
• 负责人: SANFORD J SHATTIL
• 金额: $ 40.03万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8256549
• 关键词: Address; Adhesives; Affect; Affinity; Agonist; Animal Model; Binding; Binding Proteins; Biological; Biological Models; Biology; Blood Platelets; Blood Vessels; Bone remodeling; Cardiovascular Diseases; Cell Adhesion; Cell Aggregation; Cell model; Cell physiology; Cells; Cerebral hemisphere hemorrhage; Chinese Hamster Ovary Cell; Cytoplasmic Tail; DNA Sequence Rearrangement; Defect; Development; Dorsal; Embryo; Endothelial Cells; Epithelial Cells; Event; Exhibits; Extracellular Matrix; Family; Fibrinogen; Fishes; Fluorescence; Fluorescence Resonance Energy Transfer; Follow-Up Studies; Gene Targeting; Goals; Guanosine Triphosphate Phosphohydrolases; Handedness; Heart; Hemostatic Agents; Hemostatic function; Human; Hydrocephalus; In Situ; In Vitro; Injection of therapeutic agent; Injury; Integrins; Knockout Mice; Lateral; Left; Life; Ligand Binding; Ligands; Ligation; Liver; Mediating; Mesoderm; Messenger RNA; Methods; Molecular Profiling; Monitor; Movement; Mus; Mutate; Neurologic; Oligonucleotides; Organ; Osteoclasts; Outcome; Pancreas; Phenotype; Phosphorylation; Plasma; Process; Proliferating; Proteins; Recruitment Activity; Regulation; Reporting; Signal Transduction; Signaling Protein; Smooth Muscle Myocytes; System; Tail; Talin; Techniques; Testing; Thrombin; Thrombosis; Thrombus; Tissues; Vascular Diseases; Whole Organism; Work; Zebrafish; angiogenesis; blastocyst; body asymmetry; cell motility; cell type; gastrulation; in vivo; mRNA Expression; migration; mutant; novel strategies; receptor; research study; response; src-Family Kinases; zebrafish development

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.

包括血小板在内的血管细胞表达大量整合素，双向信号传导似乎是其中大多数整合素的一般功能。虽然 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，以及它们如何向整合素传递信号或从整合素传递信号？该项目的目标是使用先进的实验方法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是由 talin 和 kindlin-3 等蛋白质向 B3 的协调募集控制的。这些蛋白质之间的二元和三元相互作用将在活细胞（包括鼠血小板）中使用双分子荧光互补、FRET 和原位邻近连接进行检查。研究将探讨 talin 和 kindlin-3 募集对 Rap1 GTPase 的依赖程度，kindlin-3 是否促进 talin 募集，反之亦然，以及与 allbB3 结合的粘附配体是否足以促进这些蛋白中的任何一个募集到 B3 尾部。第二个假设是，aV 整合素 B 胞质结构域与 talin、kindlins 和 Src 家族激酶 (SFK) 的相互作用，无论是单独还是组合，决定了体内 aV 介导过程的结果。我们对斑马鱼胚胎的初步研究使用吗啉代寡核苷酸敲低 aV，并使用 aV mRNA 来挽救敲低表型，结果表明左右体轴规范所需的原肠胚形成事件依赖于 aV，在 aV 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救援实验，以确定调节侧向性规范的相关斑马鱼整合素 aV B 亚基。为了确定是否涉及整合素与talin、kindlins或SFK的相互作用，将使用突变的p亚基进行救援实验，这些突变的p亚基被预测并被证明会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影响。