Project 1- Role of Kindlins in Blood and Vascular Cell Biology

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Project Summary Kindlin-2 (K2) is the most broadly distributed of the three mammalian kindlins. It is present in fibrobasts, chondrocytes, and cardiomocytes, and its global knockout is embryonically lethal. As shown herein, K2 is highly expressed in the three key cells of blood vessels, endothelial cells, smooth muscle cells and pericytes, where it contributes to the functional responses of these cells. Yet, our information on the role of K2 in these cells is rudimentary with many gaps in knowledge that we will seek to fill. The most well-accepted function of K2 relates to its essential role in integrin activation, but with more than 20 identified binding partners, K2 may very well connect to multiple signaling and cytoskeletal nodes. Accordingly, the hypothesis that K2's functions may be subdivided as being integrin-dependent and integrin-independent will be critically assessed. In Aim 1, this proposition will be tested by expressing either wild-type or K2Q614W615/AA (K2QW/AA) mutant, which disables its high affinity binding to integrin β subunits, and assessing specific functional responses in the three major categories of blood vessel cells. In Aim 2, the role of K2 in EC, SMC and PC will be evaluated in vivo using cell-type specific, conditional K2KO. Vascular responses to be assessed are permeability, angiogenesis, atherosclerosis and wound healing. These studies should clearly resolve the functions of K2 in the vasculature. In studies relevant to human disease, K2 expression patterns in kidneys of Systemic Lupus Erythematosus (SLE) patients and in coronary blood vessels and will be examined. In Aim 3, structure-function relationships and mechanisms by which K2 mediates cellular responses will be investigated. These studies include an effort to precisely locate two integrin-independent functions (actin and catenin binding sites in K2). Surprisingly, our structural analyses indicate that a second K2 binding site exists in the membrane proximal region (as distinguished from its well-characterized membrane distal site) in integrin β cytoplasmic tails. The function and specificity of this site in eliciting K2-dependent signaling across integrins will be investigated, and the existence and function of a homologous site for kindlin-3 in activation and signaling across αMβ2 will also be determined. Overall, these studies should establish that K2 is a master regulator of vascular cell biology and define the mechanisms underlying this role in vivo.