The Beta3 integrins play critical roles in the biological responses of cells exposed to blood. On platelets, allb33 is indispensable for platelet aggregation and thrombus formation and is a target for anti-thrombotic drugs. On endothelial cells, aV(33 is a major regulator of in angiogenesis and is also the target for drugs that suppress angiogenesis. The involvement of the two (B3 integrins in physiological and pathophysiological processes 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 a myriad of intracellular responses. Both arms of the bidirectional signaling across (B3 integrins as well as all integrins depend upon interaction of their cytoplasmic tails (CT) with binding partners. This proposal focuses on two particular binding partners of the (B3 integrins: two (Kindlin-2 and Kindlin-3) of the three member Kindlin family and one of the scr family kinases, Fyn. The hypotheses to be tested are that the two Kindlins are key regulators of the inside-out signals that lead to integrin activation and that Fyn binds differently than scr to the (B3 CT and, thereby, exerts distinct effects in controlling outside-in signaling events.
Three specific aims are proposed to test these hypotheses.
Specific Aim 1 focuses on the structure-function relationships that govern the interactions between Kindlin-2 and Kindlin-3 and B3 BT and other integrins. The ability of the Kindlins to directly induce integrin activation or to synergize with talin inside-out signaling will be examined;the mechanisms underlying the co-activator activities of the Kindlins and the structural details of the Kindlin-B3 CT interaction will be determined.
Specific Aim 2 will emphasize the effects of the Kindlins on integrin-mediated responses in platelets, megakaryocytes and endothelial cells. These analyses will involve in modulating 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.
Specific Aim 3 is predicated on the recent observation that Fyn binds to a site in the membrane proximal region of the B3 CT. The molecular details of the differential interactions of the Fyn and Scr with the B3 CT and the functional consequences of Fyn binding to this site will be determined in platelets and endothelial cells and in vivo assays of thrombosis and angiogenesis in Fyn and Scr deficient mice. Taken together, these studies will provide key insights into the way responses of the B3 integrins are initiated and propagated. This information may, in turn, establish more effective ways to design anti-thrombotic and anti-angiogenic drugs that target the B3 integrins and other integrin family members.
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