Regulation of Cell Adhesion by Affinity and Conformational Unbending of α4β1 Integrin

Chigaev, Alexandre; Waller, Anna; Zwartz, Gordon J.; Buranda, Tione; Sklar, Larry A.

doi:10.4049/jimmunol.178.11.6828

Cited by 68 publications

(128 citation statements)

References 43 publications

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“…A transition from low to high affinity state leads to additional binding of the LDV-FITC probe if cells were preincubated with the ligand at concentrations below the K d for the low affinity state and above the K d for the high affinity state. In the case of a fluorescent ligand this additional binding can be detected using a conventional flow cytometer in a homogeneous assay (4,14,15). Our present data show that binding of HUTS-21 antibodies can be used as a reporter of a ligandoccupied receptor (see Fig.…”

Section: Huts-21 Can Be Used To Detect the Activated (High Affinity) mentioning

confidence: 81%

“…2 in Ref. 4). No significant change in the rate of HUTS-21 binding was detected after cells were activated with PMA in experiments analogous to Fig.…”

Section: The Rate Of Huts-21 Binding Appears To Be Related To Inside-outmentioning

confidence: 96%

“…As shown previously, inside-out activation of integrins results in up-regulation of ligand binding affinity and unbending (extension) of the integrin molecule. The affinity change and unbending represent two different conformational changes that are regulated independently by different signaling pathways (4). Thus, we propose that an increase in the rate of HUTS-21 binding could be attributed to the unbending (extension) of the molecule induced by inside-out activa- tion.…”

Section: The Rate Of Huts-21 Binding Appears To Be Related To Inside-outmentioning

confidence: 99%

“…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.…”

Section: Methodsmentioning

confidence: 99%

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

Chigaev

Waller

Amit

et al. 2009

Journal of Biological Chemistry

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Integrins are heterodimeric adhesion receptors that regulate immune cell adhesion. Integrin-dependent adhesion is controlled by multiple conformational states that include states with different affinity to the ligand, states with various degrees of molecule unbending, and others. Affinity change and molecule unbending play major roles in the regulation of cell adhesion. The relationship between different conformational states of the integrin is unclear. Here we have used conformationally sensitive antibodies and a small LDV-containing ligand to study the role of the inside-out signaling through formyl peptide receptor and CXCR4 in the regulation of ␣ 4 ␤ 1 integrin conformation. We found that in the absence of ligand, activation by formyl peptide or SDF-1 did not result in a significant exposure of HUTS-21 epitope. Occupancy of the ligand binding pocket without cell activation was sufficient to induce epitope exposure. EC 50 for HUTS-21 binding in the presence of LDV was identical to a previously reported ligand equilibrium dissociation constant at rest and after activation. Furthermore, the rate of HUTS-21 binding was also related to the VLA-4 activation state even at saturating ligand concentration. We propose that the unbending of the integrin molecule after guanine nucleotide-binding protein-coupled receptor-induced signaling accounts for the enhanced rate of HUTS-21 binding. Taken together, current results support the existence of multiple conformational states independently regulated by both inside-out signaling and ligand binding. Our data suggest that VLA-4 integrin hybrid domain movement does not depend on the affinity state of the ligand binding pocket.In the bloodstream circulating leukocytes respond to inflammatory signals by rapid changes of cell adhesive properties.These include cell tethering, rolling, arrest, and firm adhesion, all of which are well described steps of leukocyte recruitment to the sites of inflammation (1). Leukocyte arrest and firm adhesion are mediated exclusively by integrin receptors (2). At the same time integrins can also mediate tethering and rolling (3). These largely diverse cell adhesive properties are achieved by sophisticated conformational regulation; multiple states of the same molecule with different affinity for its ligand and different degrees of molecular unbending are attributed to various types of "cellular behavior." It is proposed that the low affinity bent state translates into a non-adhesive resting cell, the low affinity unbent or extended state of integrin results in cell rolling, and the high affinity state promotes cell arrest (4, 5). However, the exact sequence of conformational events and the relationship between integrin conformational and functional activity remain key questions (6).Integrin conformation is regulated through G-protein-coupled receptors by a signaling pathway which is initiated by ligand binding to a GPCR, 3 propagated inside the cell, and results in the binding of signaling proteins (such as talin and others) to cytoplasmic domains ...

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Section: Huts-21 Can Be Used To Detect the Activated (High Affinity) mentioning

confidence: 81%

“…2 in Ref. 4). No significant change in the rate of HUTS-21 binding was detected after cells were activated with PMA in experiments analogous to Fig.…”

Section: The Rate Of Huts-21 Binding Appears To Be Related To Inside-outmentioning

confidence: 96%

Section: The Rate Of Huts-21 Binding Appears To Be Related To Inside-outmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

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

Chigaev

Waller

Amit

et al. 2009

Journal of Biological Chemistry

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“…Binding of the LDV-FITC Ligand-The VLA-4-specific ligand (LDV-FITC) has been used extensively (22,23,29) as a tool for studying VLA-4 affinity and conformation. Here we probed whether hits from screening would interfere with the binding of this ligand.…”

Section: Assay For the Detection Of Vla-4 Allosteric Antagonists-mentioning

confidence: 99%

Discovery of Very Late Antigen-4 (VLA-4, α4β1 Integrin) Allosteric Antagonists

Chigaev

Williams

et al. 2011

Journal of Biological Chemistry

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Integrins are cell adhesion receptors that mediate cell-tocell, or cell-to-extracellular matrix adhesion. They represent an attractive target for treatment of multiple diseases. Two classes of small molecule integrin inhibitors have been developed. Competitive antagonists bind directly to the integrin ligand binding pocket and thus disrupt the ligand-receptor interaction. Allosteric antagonists have been developed primarily for ␣ L ␤ 2 -integrin (LFA-1, lymphocyte function-associated antigen-1). Here we present the results of screening the Prestwick Chemical Library using a recently developed assay for the detection of ␣ 4 ␤ 1 -integrin allosteric antagonists. Secondary assays confirmed that the compounds identified: 1) do not behave like competitive (direct) antagonists; 2) decrease ligand binding affinity for VLA-4 ϳ2 orders of magnitude; 3) exhibit antagonistic properties at low temperature. In a cell based adhesion assay in vitro, the compounds rapidly disrupted cellular aggregates. In accord with reports that VLA-4 antagonists in vivo induce mobilization of hematopoietic progenitors into the peripheral blood, we found that administration of one of the compounds significantly increased the number of colony-forming units in mice. This effect was comparable to AMD3100, a well known progenitor mobilizing agent. Because all the identified compounds are structurally related, previously used, or currently marketed drugs, this result opens a range of therapeutic possibilities for VLA-4-related pathologies.

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The Ins and Outs of Integrin Signaling

Banno

Ginsberg

2008

Cell Junctions

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Regulation of Cell Adhesion by Affinity and Conformational Unbending of α4β1 Integrin

Cited by 68 publications

References 43 publications

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

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

Discovery of Very Late Antigen-4 (VLA-4, α4β1 Integrin) Allosteric Antagonists

The Ins and Outs of Integrin Signaling

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