Abstract
Integrin undergoes different activation states by changing its quaternary conformation. The integrin beta 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 alpha(L) beta2. Herein, we mapped its epitope to the critical residue Pro374 located on the inner face of the beta2 hybrid domain. Integrin alpha(L) beta2 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 alpha(L) beta2/intercellular adhesion molecule (ICAM) binding. Our study showed that Mg/EGTA-activated alpha(L)beta2 can adopt a different conformation from that activated by activating mAbs KIM185 or MEM48. Unlike ICAM-1 binding, which required only one activating agent, alpha(L) beta2/ICAM-3 binding required both Mg/EGTA and an activating mAb. This suggests that alpha(L)beta2 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 alpha(L)beta2 in the presence of Mg/EGTA, depicting an intermediate activation state, could be reverted to its resting conformation.
Highlights
Integrins represent a large family of type I heterodimeric (␣ and  subunits) membrane proteins capable of bidirectional signal transduction serving cell growth, differentiation, and apoptosis [1]
We reported the requirement of two ␣L2-activating monoclonal antibody (mAb), KIM185 and KIM127, for their adhesion to intercellular adhesion molecule (ICAM)-3 as compared with ICAM-1 [22]
Because it may serve as a useful reporter mAb for subsequent analyses of integrin affinity states, we further characterize its epitope using a panel of integrin 2 human/mouse “knock-out” mutants generated by site-directed mutagenesis [4]
Summary
Reagents and Antibodies—The following mAbs were gifts from different sources: MHM24 (anti-␣L) and MHM23 (anti-2, heterodimer dependent) were obtained from Prof. Thereafter, cells were incubated in Mg/EGTA (5 mM MgCl2 and 1.5 mM EGTA) or phorbol 12-myristate 13-acetate (PMA) (100 ng/ml) with mAbs (irrelevant mAb, MHM23, or MEM148) in RPMI media containing 5% (v/v) heat-inactivated fetal bovine serum and 10 mM HEPES (pH 7.4) for 30 min at 37 °C. Cells labeled with 3.0 mM 2Ј7Ј-bis-(2-carboxyethyl)-5-(and-6) carboxyfluorescein fluorescent dye (Molecular Probes, Eugene, OR) were incubated in RPMI wash buffer containing Mg/EGTA (5 mM MgCl2 and 1.5 mM EGTA) and/or activating mAb (10 g/ml) to activate ␣L2-mediated adhesion. Soluble ICAMs were prepared by incubating 20 g/ml ICAM-Fc with 100 g/ml FITC-conjugated rabbit anti-human IgG Fc antibody (Pierce) in 50 l of RPMI 1640 media containing 5% (v/v) heat-inactivated fetal bovine serum and 10 mM HEPES (pH 7.4) for 30 min at room temperature. Function-blocking ␣L-specific mAb MHM24 (10 g/ml) was included in the samples
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