The ?3 integrins play critical roles in the biological responses of cells exposed to blood. On platelets, aIIb?3 is indispensable for platelet aggregation and thrombus formation. On endothelial cells, av?3 is a major regulator of the adhesive and migratory responses of the cells and influences angiogenesis. The involvement of the ?3 integrins in biological responses depends upon signaling across the integrins;inside- out signaling to control the activation state of the integrins and outside-in signaling to control cytoskeletal connections and intracellular signaling. Both arms of the bidirectional signaling across ?3 integrins as well as all integrins depend upon interaction of their short cytoplasmic tails (CT) with binding partners. This proposal focuses on a newly recognized family of binding partners of the ?3 CT, the kindlins, and particularly on their role in integrin activation. Very recent publications, including ones from the applicant's laboratory, suggest a pivotal role of kindlin family members in the function of aIIb?3 and av?3. The primary hypothesis to be tested is that kindlin-2 and kindlin-3 regulate integrin activation. Mechanistically, it is postulated that this activity relies on binding of the kindlins to the membrane distal region of the ?3 CT where each can cooperate with a second ?3 CT binding partner, talin.
Three specific aims are proposed.
Aim 1 focuses on the requirements and functional outcomes of kindlin:integrin interaction. The selectivity of kindlin binding to integrin 2 subunits will be examined;the ability of the two kindlins to directly induce integrin activation or synergize with talin in inside-out signaling will be determined;and the structure that the ?3 CT assumes when complexed with kindlins will be solved.
Aim 2 addresses the mechanisms underlying the co-activator activities of kindlins and considers how phosphorylation of the ?3 CT influence kindlin binding.
Aim 3 emphasizes the effects of the kindlins on integrin-mediated responses in intact cells, platelets, megakaryocytes and endothelial cells. These analyses will involve modulation of kindlin levels and functions in these cells using siRNA and membrane permeable peptides. The effects of reduced levels of kindlin-2 in vivo will also be analyzed in mice. Taken together, these studies will provide key insights into the way biological responses of the ?3 integrins are initiated. This information may, in turn, establish more effective ways to design anti-thrombotic and anti-angiogenic drugs that target the ?3 integrins and other integrin family members.
Platelet aggregation is essential for thrombus formation and, hence, underlies the thrombotic diseases of heart attack and stroke. Similarly, angiogenesis, the formation of new blood vessels, is involved in cancer and the salvage of damaged heart tissue. We seek to define how a new set of molecules influence platelets and endothelial cells to control aggregation and angiogenesis, thereby identifying new targets for treatment and prevention of cardiovascular disease.
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