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J. Biol. Chem., Vol. 280, Issue 32, 29208-29216, August 12, 2005

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Epitope Mapping of Monoclonal Antibody to Integrin _{L 2} Hybrid Domain Suggests Different Requirements of Affinity States for Intercellular Adhesion Molecules (ICAM)-1 and ICAM-3 Binding^*

Ren-Hong Tang, Emilia Tng, S. K. Alex Law, and Suet-Mien Tan¶

From the School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 and Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom

Integrin undergoes different activation states by changing its quaternary conformation. The integrin 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 _{L 2}. Herein, we mapped its epitope to the critical residue Pro³⁷⁴ located on the inner face of the ₂ hybrid domain. Integrin _{L 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 _{L 2}/intercellular adhesion molecule (ICAM) binding. Our study showed that Mg/EGTA-activated _L2 can adopt a different conformation from that activated by activating mAbs KIM185 or MEM48. Unlike ICAM-1 binding, which required only one activating agent, _{L 2}/ICAM-3 binding required both Mg/EGTA and an activating mAb. This suggests that _L2 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 _L2 in the presence of Mg/EGTA, depicting an intermediate activation state, could be reverted to its resting conformation.

Received for publication, March 24, 2005 , and in revised form, May 31, 2005.

^* This work was supported by Nanyang Technological University Grant M52080022 and Agency for Science and Technology (A^*STAR) Biomedical Research Council Grants 03/1/22/18/219 and 04/1/22/19/358. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 65-6-3162834; Fax: 65-6-7913856; E-mail: smtan{at}ntu.edu.sg.

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