Abstract 496: Competition Between Filamin and Kindlin-2 Regulates Integrin Activation

Abstract

Cell migration and adhesion during hemostasis, angiogenesis and inflammation are dynamically regulated by integrin heterodimeric adhesion receptors. Their interactions with cytosolic proteins, filamin (FLN), talin (TLN) and Kindlin (Kn2) enable them to convey intracellular signals (inside-out-signaling) to the external environment by engaging extracellular matrix ligands. While TLN and Kn2 activate integrins, FLN inhibits cell migration. TLN and Kn2 bind to membrane-proximal and -distal NPxY motifs of β integrin cytoplasmic tails (CTs), respectively, and an integrin binding site for FLN resides in between these two sequences. Competition between TLN and FLN regulates integrin activation, but it is unknown if FLN and Kn2 compete and regulate integrin inside-out signaling. This competition was tested using αIIbβ3 (platelet-specific) and β7 (lymphocyte-specific; strong FLN binder) integrins in multiple cell types. siRNA depletion of FLNA in K562 cells stably expressing αIIbβ3 integrin (K562-αIIbβ3) significantly enhanced PAC-1 (specific for activated αIIbβ3) binding compared to control siRNA, demonstrating its effect on β3 activation. In pulldown assays using GST-β3 CT, Kn2 bound β3 in CHO lysates transfected with Kn2, either alone or with FLN repeat 21; however, FLN binding to β3 CT was observed only when FLN repeat 21 was expressed alone. Under similar conditions using GST-β7 CT, FLN-β7 interaction was not perturbed by Kn2. This was more pronounced in endothelial cell lysates where GST-β7 CT bound endogenous FLNA but not Kn2. Weak talin-β7 CT binding in this assay was noted. Moreover, in K562-αIIbβ3 cells, exogenous Kn2 overcame the suppressive effect of FLN on αIIbβ3 activation. Overall, our data shows that FLN inhibits β3 integrin function, and competition between FLN and Kn2 can indeed regulate integrin activation.