Abstract 369: Localization and Specification of a Kindlin-2 Binding Core Within Integrin Beta Cytoplasmic Tails
Integrins are widely expressed cell adhesion receptors. One of the key regulatory mechanisms of integrins is their ability to undergo activation. Based on in vivo studies kindlins and talin are know to play indispensable roles in integrin activation, and this function depends on their binding to the cytoplasmic tail (CT) of integrin β subunits, However, the requisite sequences within the integrin β subunit that specify kindlin is as of yet unresolved.
Using overlapping synthetic peptides corresponding to the highly conserved C-terminal region of integrin β CT as inhibitors of β3 CT: kindlin-2 interaction in pull-down assays, we identified the kindlin-2 binding core to a twelve amino-acid sequence. This sequence includes the membrane-distal NxxY region and S/T cluster, which are conserved across integrin β CT, but also variable residues flanking these two motifs. We postulated that the contribution of these flanking residues in β subunits might impose selectivity upon the interaction of kindlin-2 with various β subunits. Indeed, we found that kindlin-2 interacts with β1and β3, but poorly with the β2 CT, in both pull-down and surface plasmon resonance experiments. This selectivity was determined primarily by the non-conserved residues at the extreme C-termini of the β CT. By swapping amino acids in this region, reactivity with kindlin-2 could either be enhanced or blunted. By surface plasmon resonance, we also demonstrated that the β3 subunits can accommodate both kindlin-2 and talin head, even though their binding sites are very close in the β3 subunit. Kindlin-2 and talin head do not interact with each other but can bind simultaneously to the integrin β3 CT without mutual disturbance. This work specifies a binding site within β CT for kindlin-2, provides the first evidence for selectivity in the interaction of kindlin-2 with integrin β subunits, and excludes a spatial influence of talin and kindlin-2 on their binding to integrin CT in supporting integrin activation.
- © 2012 by American Heart Association, Inc.