Disruption of the integrin αLβ2 transmembrane domain interface by β2 Thr-686 mutation activates αLβ2 and promotes micro-clustering of the αL subunits

Integrins are type I heterodimeric cell adhesion molecules that mediate a wide array of biological processes. Integrin bidirectional signaling allows communication between the cell interior with its microenvironment. The integrin transmembrane domains (TMs) are the transducers of activation signal that is relayed from the cytoplasmic domains to the distal ligand binding site located in the ectodomain of the integrin and vice versa. In this study, we showed that the disruption of the αLβ2 TMs by mutation of a key interface residue Thr-686 in the β2 TM promoted αLβ2 activation with ICAMs binding properties that are reminiscent of an intermediate affinity receptor. The activated αLβ2 TM mutants, however, showed minimal reactivity with the reporter mAb KIM127 that recognizes a highly extended αLβ2. Two models of αLβ2 TM interaction were proposed previously. One with GXXXG-type interaction, and another that is based on TM cysteine-scanning analyses. Our data are consistent with a GXXXG-type interaction of the αLβ2 TMs. Finally, we observed by FRET analyses that perturbation of the αLβ2 TMs by β2 Thr-686 mutation facilitated αL microcluster formation. This was diminished by linking the αLβ2 TMs with a disulfide bond, which served to clasp the TMs. These data suggest that disruption of the TM interface changes αLβ2 ligand binding affinity, and it may contribute to αL micro-cluster formation.