Cytoskeletal Control of Beta-2 Integrin Conformational Change During Leukocyte Activation

b₂ integrin molecules are maintained in an inactive form in resting leukocytes by constraints from the cytoskeletal complex. It is widely accepted that the inside-out signals relax the cytoskeletal constraints to facilitate integrin clustering and conformational changes that provide high avidity and affinity binding to the ligand. Objectives: To study the roles of the cytoskeleton in the b₂ integrin’s conformational change and in the outside-in signaling induced by this change. Methods: We used divalent cations (Mn⁺⁺ ) to induce conformational changes of b₂ integrin to a high affinity state and detected the molecular mobility of the high-affinity state integrin using the single particle tracking (SPT) method. Results: We found that integrin molecular mobility in cells treated with 500nM Mn⁺⁺ increased 4-5 fold over the control cells, demonstrating that b₂ integrin molecules broke away from the cytoskeleton constraint during Mn⁺⁺ induced conformational change. Further experiments revealed that this breaking away from the cytoskeleton was accompanied by an increase in tyrosine phosphorylation of paxillin, a hallmark of b₂ integrin-induced outside-in signaling. When paxillin phosphorylation was inhibited by genistein, the increased mobility of b₂ integrin was not affected. This indicated cytoskeleton relaxation was upstream of paxillin phosphorylation. To rule out any possible interference from inside-out pathways, PKC inhibitor was used, and it did not block cytoskeletal relaxation during Mn⁺⁺ -induced conformational change. Conclusion: These results indicated that b₂ integrin conformational change from resting to activated state resulted in b₂ integrin relaxation from cytoskeletal constraint and also strongly suggested that cytoskeletal relaxation is an early event in the outside-in signal transduction pathway.

This work is supported by NIH: GM54715 and AHA: 0240017N