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J. Biol. Chem., Vol. 279, Issue 31, 32435-32443, July 30, 2004

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Conformational Regulation of ₄₁-Integrin Affinity by Reducing Agents

"INSIDE-OUT" SIGNALING IS INDEPENDENT OF AND ADDITIVE TO REDUCTION-REGULATED INTEGRIN ACTIVATION^*

Alexandre Chigaev, Gordon J. Zwartz, Tione Buranda, Bruce S. Edwards, Eric R. Prossnitz, and Larry A. Sklar¶||

From the Department of Pathology and the Cancer Center and the Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131 and the ^¶National Flow Cytometry Resource, Los Alamos National Laboratory, Los Alamos, New Mexico 87545

The ₄₁-integrin (very late antigen-4 (VLA-4), CD49d/CD29) is an adhesion receptor involved in the interaction of lymphocytes, dendritic cells, and stem cells with the extracellular matrix and endothelial cells. This and other integrins have the ability to regulate their affinity for ligands through a process termed "inside-out" signaling that affects cell adhesion avidity. Several mechanisms are known to regulate integrin affinity and conformation: conformational changes induced by separation of the C-terminal tails, divalent ions, and reducing agents. Recently, we described a fluorescent LDV-containing small molecule that was used to monitor VLA-4 affinity changes in live cells (Chigaev, A., Blenc, A. M., Braaten, J. V., Kumaraswamy, N., Kepley, C. L., Andrews, R. P., Oliver, J. M., Edwards, B. S., Prossnitz, E. R., Larson, R. S., and Sklar, L. A. (2001) J. Biol. Chem. 276, 48670–48678). Using the same molecule, we also developed a fluorescence resonance energy transfer-based assay to probe the "switchblade-like" opening of VLA-4 upon activation. Here, we investigated the effect of reducing agents on the affinity and conformational state of the VLA-4 integrin simultaneously with cell activation initiated by inside-out signaling through G protein-coupled receptors or Mn²⁺ in live cells in real time. We found that reducing agents (dithiothreitol and 2,3-dimercapto-1-propanesulfonic acid) induced multiple states of high affinity of VLA-4, where the affinity change was accompanied by an extension of the integrin molecule. Bacitracin, an inhibitor of the reductive function of the plasma membrane, diminished the effect of dithiothreitol, but had no effect on inside-out signaling. Based on this result and differences in the kinetics of integrin activation, we conclude that conformational activation of VLA-4 by inside-out signaling is independent of and additive to reduction-regulated integrin activation.

Received for publication, April 21, 2004

^* This work was supported by National Institutes of Health Grants P50 HL56384, IR01EB02022, and IR24 CA88339 (to L. A. S.). 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.

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Fig. 1.

^|| To whom correspondence and reprint requests should be addressed: Dept. of Pathology and Cancer Center, University of New Mexico HSC, Albuquerque, NM 87131. Tel.: 505-272-6892; Fax: 505-272-6995; E-mail: lsklar{at}salud.unm.edu.

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