Kindlins, Integrin Activation and the Regulation of Talin Recruitment to αIIbβ3

Talins and kindlins bind to the integrin beta 3 cytoplasmic tail and both are required for effective activation of integrin alpha IIb beta 3 and resulting high-affinity ligand binding in platelets. However, binding of the talin head domain alone to b3 is sufficient to activate purified integrin alpha IIb beta 3 in vitro. Since talin is localized to the cytoplasm of unstimulated platelets, its re-localization to the plasma membrane and to the integrin is required for activation. Here we explored the mechanism whereby kindlins function as integrin co-activators. To test whether kindlins regulate talin recruitment to plasma membranes and to alpha IIb beta 3, full-length talin and kindlin recruitment to b3 was studied using a reconstructed CHO cell model system that recapitulates agonist-induced alpha IIb beta 3 activation. Over-expression of kindlin-2, the endogenous kindlin isoform in CHO cells, promoted PAR1-mediated and talin-dependent ligand binding. In contrast, shRNA knockdown of kindlin-2 inhibited ligand binding. However, depletion of kindlin-2 by shRNA did not affect talin recruitment to the plasma membrane, as assessed by subcellular fractionation, and neither over-expression of kindlins nor depletion of kindlin-2 affected talin interaction with alpha IIb beta 3 in living cells, as monitored by bimolecular fluorescence complementation. Furthermore, talin failed to promote kindlin-2 association with alpha IIb beta 3 in CHO cells. In addition, purified talin and kindlin-3, the kindlin isoform expressed in platelets, failed to promote each other's binding to the b3 cytoplasmic tail in vitro. Thus, kindlins do not promote initial talin recruitment to alpha IIb beta 3, suggesting that they co-activate integrin through a mechanism independent of recruitment.