Probing Conformational Changes in the I-like Domain and the Cysteine-rich Repeat of Human β3 Integrins following Disulfide Bond Disruption by Cysteine Mutations

Chen, Ping; Melchior, Chantal; Brons, Nicolaas H. C.; Schlegel, Nicole; Caen, Jacques P.; Kieffer, Nelly

doi:10.1074/jbc.m105737200

Cited by 62 publications

(59 citation statements)

References 47 publications

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“…Previous studies showed that integrin conformation may be altered by mutations or redox changes on the cysteine residues of their subunit peptides [10][11][12][13][14][15][16][17]. Here, we investigated how VLA-4 integrin ligand binding activity can be regulated by redox modulation.…”

Section: Introductionmentioning

confidence: 99%

Ligand binding of leukocyte integrin very late antigen‐4 involves exposure of sulfhydryl groups and is subject to redox modulation

et al. 2008

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Activation of leukocyte integrins is important for selective recruitment of cells from the circulation to tissues. Our previous studies showed that the binding between the integrin very late antigen-4 (VLA-4) and vascular cell adhesion molecule-1 (VCAM-1) is modulated by reactive oxygen species. In this study, we investigated the molecular nature of redox modulation on the activation states of VLA-4 on human leukocytes. We found that ligand binding of VLA-4 induced exposure of sulfhydryl groups on the a4 peptide. Low concentrations (5-10 lM) of exogenous hydrogen peroxide in the presence or absence of added glutathione enhanced the ligand binding ability of VLA-4 to VCAM-1 and cell rolling on VCAM-1, while higher concentrations ( ! 100 lM) of hydrogen peroxide inhibited the binding. Exogenous hydrogen peroxide and glutathione induced molecular modification of S-glutathionylation on the a4 peptide. The redox regulation of the VLA-4 binding activity required outside-in signaling and cytoskeleton rearrangement. Our results indicate that ligand binding of VLA-4 involves redox modulations which may play a pivotal role in regulating the activation states of VLA-4 in inflammatory tissues and hence direct leukocyte trafficking.

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Section: Introductionmentioning

confidence: 99%

Ligand binding of leukocyte integrin very late antigen‐4 involves exposure of sulfhydryl groups and is subject to redox modulation

et al. 2008

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“…Owing to a cohort of point mutations affecting integrin function, the αIIbβ 3 -integrin is one of the best genetically analysed integrins. Point mutations and deletions that affect αIIb-and β 3 -integrin function have been mapped to the cytoplasmic as well as ligand-binding and extracellular linker domains, giving additional relevance to the allosteric model implicating all different regions of the αβ-heterodimer [41][42][43][44]. Similar to LAD-1, αIIbβ 3 -integrin activation and clustering have to be very rapid to form a functional blood clot.…”

Section: Glanzmann Thrombastheniamentioning

confidence: 99%

Integrin‐dependent pathologies

Wehrle-Haller¹,

Imhof²

2003

The Journal of Pathology

103

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Cell adhesion and migration are essential for embryonic development, tissue regeneration, and immune defence. The physical link between the extracellular substrate and the actin cytoskeleton is mediated by receptors of the integrin family and a large set of adaptor proteins. During cell migration this physical link is dynamically modified, allowing the cell to sense and adapt to the microenvironment. This includes the formation of integrin clusters at the cell front, their stabilization in the cell body and subsequent disassembly of these clusters at the rear of the cell. The modulation of the adhesion strength of the cell to the substrate is regulated by the affinity switch of integrin molecules and increased avidity through clustering of integrins. Here we explain how integrins mediate cell migration and how genetic defects of integrins and their adaptors lead to cellular dysfunction and generate pathological situations.

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“…Integrins: Three or More Independent Activating Mechanisms-Integrin conformational change and activation can be achieved under different conditions: divalent ions, reducing agents, inside-out signaling, mutations in extracellular domains and C-terminal tails, and disulfide disruption (11,23,25,26,43). Several opposing mechanisms may contribute to integrin conformational change with divalent ions.…”

Section: Kinetics Of Dtt-induced Extension Detected Using Fret Coincimentioning

confidence: 99%

“…A number of mutational studies have shown that disruption of disulfides in the integrin ␤ 3 -subunit results in constitutively active integrins (Cys 5 , Cys 435 , Cys 560 , Cys 598 , Cys 663 , and Cys 687 ) (25)(26)(27)(28). Truncated ␤ 3 -subunits (amino acids 1-469) lacking the Cys-rich domain form heterodimers that bind fibrinogen with high affinity (29).…”

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confidence: 99%

Conformational Regulation of α4β1-Integrin Affinity by Reducing Agents

Chigaev¹,

Zwartz²,

Buranda³

et al. 2004

Journal of Biological Chemistry

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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,...

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Probing Conformational Changes in the I-like Domain and the Cysteine-rich Repeat of Human β3 Integrins following Disulfide Bond Disruption by Cysteine Mutations

Cited by 62 publications

References 47 publications

Ligand binding of leukocyte integrin very late antigen‐4 involves exposure of sulfhydryl groups and is subject to redox modulation

Ligand binding of leukocyte integrin very late antigen‐4 involves exposure of sulfhydryl groups and is subject to redox modulation

Integrin‐dependent pathologies

Conformational Regulation of α4β1-Integrin Affinity by Reducing Agents

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