Epitope mapping of monoclonal antibody to integrin α Lβ2 hybrid domain suggests different requirements of affinity states for intercellular adhesion molecules (ICAM)-1 and ICAM-3 binding

Abstract

Integrin undergoes different activation states by changing its quaternary conformation. The integrin β hybrid domain acts as a lever for the transmission of activation signal. The displacement of the hybrid domain can serve to report different integrin activation states. The monoclonal antibody (mAb) MEM148 is a reporter antibody that recognizes Mg/EGTA-activated but not resting integrin αLβ2. Herein, we mapped its epitope to the critical residue Pro374 located on the inner face of the β2 hybrid domain. Integrin αLβ 2 binds to its ligands ICAM-1 and ICAM-3 with different affinities. Integrin is proposed to have at least three affinity states, and the position of the hybrid domain differs in each. We made use of the property of mAb MEM148 to analyze and correlate these affinity states in regard to α Lβ2/intercellular adhesion molecule (ICAM) binding. Our study showed that Mg/EGTA-activated αLβ2 can adopt a different conformation from that activated by activating mAbs KIM185 or MEM48. Unlike ICAM-1 binding, which required only one activating agent, αLβ2/ICAM-3 binding required both Mg/EGTA and an activating mAb. This suggests that αLβ2 with intermediate affinity is sufficient to bind ICAM-1 but not ICAM-3, which requires a high affinity state. Furthermore, we showed that the conformation adopted by αLβ2 in the presence of Mg/EGTA, depicting an intermediate activation state, could be reverted to its resting conformation. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.