This proposal will continue the atomic level investigation of integrin activation - a central response for all integrin-mediated cell adhesive processes. Discovered more than two decades ago, integrins have been widely recognized as major cell surface receptors that mediate a variety of cellular processes including cellextracellular matrix (ECM) adhesion, cell migration, cell shape change, and cell survival. Integrin activation occurs via a distinct """"""""inside-out"""""""" signaling process in which the integrin cytoplasmic face first senses a conformational signal that relays through the transmembrane region to the extracellular domain, thus converting the receptor from a low to a high affinity state. Over the years, our laboratory and many others have attempted to understand the molecular details of this inside-out activation process. Using NMR spectroscopy as a major tool, combined with collaborative functional approaches, we have been focusing on studying platelet allb(33 - a prototypic integrin that plays a key role in hemostasis and thrombosis. We have shown in a series of studies that the allb/p3 cytoplasmic tails (CTs) of this receptor can undergo clasping/unclasping process, thus promoting the integrin inside-out activation. We have further shown that the unclasping process of integrin allbps is triggered by talin - a major cytoskeletal adaptor that has been established as the essential component of the integrin activation. Our most recent data have indicated that the activity of talin is also conformationally regulated. Our findings have led to a comprehensive model for integrin activation where a series of energy-dependent conformational changes need to occur on the integrin intracellular side to initiate the integrin transmembrane signaling and its high affinity ligand binding. In this continuation proposal, we will vigorously test this model by asking the following questions: (i) How does the change of the integrin intracellular face propagate to its transmembrane domain, a central region that connects the intracellular and the extracellular sides of the receptor? While 3D structures of both extracellular and intracellular domains of integrins have been reported, an atomic view of this integrin central piece is still lacking, (ii) What is the atomic basis of the talin authoinhibition and how is it activated and regulated to trigger the integrin inside-out signaling? The answer to these questions is vital for a thorough understanding of the integrin function and is also fundamental for cell biology and signal transduction. We will continue to use NMR spectroscopy as a core technique to address these questions. In continued collaboration with Ed Plow and other project leaders, we will perform various functional experiments to corroborate our NMR-based findings. Our results, if successful, will lead to another significant advance for understanding the integrin signaling. They will also promote the understanding and treatment of allb|33-mediated diseases such as thrombosis and atherosclerosis.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Program Projects (P01)
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Heart, Lung, and Blood Initial Review Group (HLBP)
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Cleveland Clinic Lerner
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Bledzka, Kamila; Bialkowska, Katarzyna; Sossey-Alaoui, Khalid et al. (2016) Kindlin-2 directly binds actin and regulates integrin outside-in signaling. J Cell Biol 213:97-108
Plow, Edward F; Das, Mitali; Bialkowska, Katarzyna et al. (2016) Of Kindlins and Cancer. Discoveries (Craiova) 4:
Sossey-Alaoui, Khalid; Plow, Edward F (2016) miR-138-Mediated Regulation of KINDLIN-2 Expression Modulates Sensitivity to Chemotherapeutics. Mol Cancer Res 14:228-38
Biswas, Sudipta; Xin, Liang; Panigrahi, Soumya et al. (2016) Novel phosphatidylethanolamine derivatives accumulate in circulation in hyperlipidemic ApoE-/- mice and activate platelets via TLR2. Blood 127:2618-29
Plow, Edward F (2016) The why's and wherefore's of this vascular biology section of Current Opinion in Hematology. Curr Opin Hematol 23:233-4
Meller, Julia; Rogozin, Igor B; Poliakov, Eugenia et al. (2015) Emergence and subsequent functional specialization of kindlins during evolution of cell adhesiveness. Mol Biol Cell 26:786-96
Liu, Jianmin; Das, Mitali; Yang, Jun et al. (2015) Structural mechanism of integrin inactivation by filamin. Nat Struct Mol Biol 22:383-9
Plow, Edward F; Qin, Jun (2015) The role of RIAM in platelets put to a test. Blood 125:207-8
Bialkowska, Katarzyna; Byzova, Tatiana V; Plow, Edward F (2015) Site-specific phosphorylation of kindlin-3 protein regulates its capacity to control cellular responses mediated by integrin αIIbβ3. J Biol Chem 290:6226-42
Xu, Zhen; Chen, Xue; Zhi, Huiying et al. (2014) Direct interaction of kindlin-3 with integrin αIIbβ3 in platelets is required for supporting arterial thrombosis in mice. Arterioscler Thromb Vasc Biol 34:1961-7

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