Conformational Regulation of α4β1-Integrin Affinity by Reducing Agents

Abstract

The ␣ 4 ␤ 1 -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 LDVcontaining 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 proteincoupled receptors or Mn 2؉ 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.The ␣ 4 ␤ 1 -integrin (very late antigen-4 (VLA-4), 1 CD49d/ CD29) is a heterodimeric protein and a member of the family of adhesion receptors that is broadly expressed in lymphocytes and dendritic cells (1) and stem cells (2). VLA-4 has a flexible molecular structure that allows initial capture, tethering, rolling, and firm attachment of cells using the same counterstructure (3, 4). These properties appear to result from regulation of affinity and conformation by "inside-out" signaling (5, 6). Although the precise molecular mechanism of integrin conformational activation is unknown, significant understanding of this mechanism has been achieved in the last several years.One model involves the "mechano-conformational" regulation of integrin affinity and conformation. It is based on the idea that the separation of the intracellular ␣-and ␤-subunit tails may initiate "a piston-like or scissors-like motion" of the transmembrane domains (7). This motion results in a large conformational rearrangement of the integrin, accompanied by a "switchblade-like" opening of the molecule (8) and its conformational activation (5,...

Conformational changes in human integrin αIIbβ3 after platelet activation, monitored by FRET

“…The FRET efficiency results (Table 3) Our findings are consistent with other observations such as the capacity of the bent conformation of the α V β 3 full-length ectodomain to stably bind RGD peptides and a fragment of fibronectin (domains 7 to 10 and EDB), as found by X-ray crystallography [3] and electron microscopy [8], respectively. Thus, these and our observations are neither consistent with the fully extended structure of the soluble full-length α V β 3 ectodomain in the presence of Mn 2+ or Mn 2+ + a cyclic RGD peptide, as observed by electron microscopy [7] nor with the interpretation of energy transfer experiments on Mn 2+ and chemokine-activated α 4 β 1 integrin in U937 monoblastoid cells and in U937 cells transfected with the formil peptide receptor [13,42]. The discrepancies with Takagi et al [7] may come from the significant modifications introduced in the recombinant full-length ectodomain they used and from the image selection and data processing methods they employed, as already pointed out by Adair et al [8].…”

Real-time Analysis of Conformation-sensitive Antibody Binding Provides New Insights into Integrin Conformational Regulation

“…In the presence of a high concentration of LDV-FITC (100 nM), an unquenching of the fluorescence resonance energy transfer signal, which is interpreted as molecular unbending, can be detected after activation thorough several GPCRs, Mn 2ϩ , reducing agents, Ca 2ϩ ionophore, etc. (4,16,33). At the same time, the low affinity state of VLA-4 (or at least the lowest affinity state that we have ever seen) can be detected on resting U937 cells even in the presence of a saturating amount of LDV ligand.…”

“…Activation of U937 cells through a non-desensitizing mutant of FPR results in the high affinity state of the VLA-4 ligand binding pocket, which persists for more than 1000 s (4,14,33). In this case the absence of a major HUTS-21 epitope exposure suggests that hybrid domain swing-out is not directly related to the induction of the high affinity state.…”

“…Inside-out activation induces the high affinity and unbent (extended) state of VLA-4 (4,14,16,33). However, without ligand present, inside-out activation did not result in a major exposure of the HUTS-21 epitope (Fig.…”

“…After activation by formyl peptide, the affinity state is up-regulated, and rapid molecule unbending (extension) can also be detected using a fluorescence resonance energy transfer-based assay. This activated state persists for more than 25-30 min because of the lack of receptor desensitization (4,14,16,33). We took advantage of this well characterized model system to study how HUTS-21 would report this persistent activated state.…”