Integrin Activation by Dithiothreitol or Mn2+ Induces a Ligand-occupied Conformation and Exposure of a Novel NH2-terminal Regulatory Site on the β1Integrin Chain

Self Cite

110

It was originally thought that the critical role of the CD40 ligand (CD40L) in normal and inflammatory immune responses was mainly mediated through its interaction with the classic receptor, CD40. However, data from CD40L ؊/؊ and CD40؊/؊ mice suggest that the CD40L-induced inflammatory immune response involves at least one other receptor. This hypothesis is supported by the fact that CD40L stabilizes arterial thrombi through an ␣IIb␤3-dependent mechanism. Here we provide evidence that soluble CD40L (sCD40L) binds to cells of the undifferentiated human monocytic U937 cell line in a CD40-and ␣IIb␤3-independent manner. Binding of sCD40L to U937 cells was inhibited by anti-CD40L monoclonal antibody 5C8, anti-␣5␤1 monoclonal antibody P1D6, and soluble ␣5␤1. The direct binding of sCD40L to purified ␣5␤1 was confirmed in a solid phase binding assay. Binding of sCD40L to ␣5␤1 was modulated by the form of ␣5␤1 expressed on the cell surface as the activation of ␣5␤1 by Mn 2؉ or dithiothreitol resulted in the loss of sCD40L binding. Moreover, sCD40L induced the translocation of ␣5␤1 to the Triton X-100-insoluble fraction of U937 cells, the rapid activation of the MAPK pathways ERK1/2, and interleukin-8 gene expression. The binding of sCD40L to CD40 on BJAB cells, an ␣5␤1-negative B cell line, and the resulting activation of ERK1/2 was not inhibited by soluble ␣5␤1, suggesting that sCD40L can bind concomitantly to both receptors. These results document the existence of novel CD40L-dependent pathways of physiological relevance for cells expressing multiple receptors (CD40, ␣5␤1, and ␣IIb␤3) for CD40L. (2). The expression of CD154 is inducible, and its expression on T cells is triggered primarily by T cell receptor signaling and is regulated by CD28-dependent and independent pathways (3) CD40L is stored in platelets and a subpopulation of T cells and rapidly translocates to the cell membrane following T cell and platelet activation (4, 5). A soluble form of biologically active CD40L trimer (sCD40L) is present in the supernatant of activated T cells (6) and platelets (7) and results from the proteolytic cleavage of the homotrimeric CD40L by a metalloproteinase (7).It was originally thought that CD40L had only one receptor, CD40, which is a type I transmembrane protein that is a member of the TNF receptor superfamily. CD40 is expressed on the surface of many immune and non-immune cells, including B lymphocytes, monocytes/macrophages, and dendritic cells, as well as platelets, epithelial, and endothelial cells (8). Most biological functions of CD40L have been attributed to its direct interaction with CD40. However, studies using CD40L Ϫ/Ϫ and CD40 Ϫ/Ϫ mice have suggested that CD40L may also bind to one or more other receptors (9). In support of this hypothesis, it has been elegantly demonstrated that sCD40L interacts with ␣IIb␤3 (GPIIb/IIIa), an integrin expressed on platelets (10), triggering outside-in signaling and inducing platelet activation and spreading (11). CD40L Ϫ/Ϫ mice exhibit increased bleeding time (12) and reduce...

“…4). Second, conformational changes induced by these chemical agents expose a ␤1 epitope, the mAb B44 epitope (21). Indeed, the results presented in Fig.…”

Section: Resultsmentioning

confidence: 86%

CD40 Ligand Binds to α5β1 Integrin and Triggers Cell Signaling

Léveillé

Bouillon

Wen

et al. 2007

Self Cite

110

“…Similarly, a possible explanation to the increased adhesion of IL-5-treated EOS to 7d-VCAM-1 under flow after preincubation with anti-␣M is that the mAb, when interacting with the IL-5-induced conformation of ␣M␤2, enhances ␣M␤2-mediated trans-regulation and stimulates ␣4␤1-mediated adhesion. In the case of the ␤1 subunit, a 12-amino acid sequence in the ␤1 headpiece is recognized by both inhibitory and stimulatory mAbs (74), indicating that there is a fine line between inhibition and activation of integrins.…”

Section: Discussionmentioning

confidence: 99%

Differential Engagement of Modules 1 and 4 of Vascular Cell Adhesion Molecule-1 (CD106) by Integrins α4β1 (CD49d/29) and αMβ2 (CD11b/18) of Eosinophils

Barthel

Annis

Mosher

et al. 2006

We have studied adhesion of eosinophils to various forms of vascular cell adhesion molecule 1 (VCAM-1, CD106), an integrin counter-receptor implicated in eosinophil recruitment to the airway in asthma. Full-length 7d-VCAM-1, with seven immunoglobulin-like modules, contains integrin-binding sites in modules 1 and 4. The alternatively spliced six-module protein, 6d-VCAM-1, lacks module 4. In static assays, unactivated purified human blood eosinophils adhered similarly to recombinant soluble human 6d-VCAM-1 and 7d-VCAM-1 coated onto polystyrene microtiter wells. Further experiments, however, revealed differences in recognition of modules 1 and 4. Antibody blocking indicated that eosinophil adhesion to 6d-VCAM-1 or a VCAM-1 construct containing only modules 1-3, 1-3VCAM-1, is mediated by ␣4␤1 (CD49d/29) ,whereas adhesion to a construct containing modules 4 -7, 4 -7VCAM-1, is mediated by both ␣4␤1 and ␣M␤2 (CD11b/18). Inhibitors of phosphoinositide 3-kinase, which block adhesion of eosinophils mediated by ␣M␤2, blocked adhesion to 4 -7VCAM-1 but had no effect on adhesion to 6d-VCAM-1. Consistent with the antibody and pharmacological blocking experiments, eosinophilic leukemic cell lines lacking ␣M␤2 did not adhere to 4 -7VCAM-1 but did adhere to 6d-VCAM-1 or 1-3VCAM-1. Activation of eosinophils by interleukin (IL)-5 enhanced static adhesion to 6d-VCAM-1, 7d-VCAM-1, or 4 -7VCAM-1; IL-5-enhanced adhesion to all 3 constructs was blocked by anti-␣M␤2. Adhesion of unstimulated eosinophils to 7d-VCAM-1 under flow conditions was inhibited by anti-␣4 or anti-␣M. IL-5 treatment decreased eosinophil adhesion to 7d-VCAM-1 under flow, and anti-␣M had the paradoxical effect of increasing adhesion. These results demonstrate that ␣M␤2 modulates ␣4␤1-mediated eosinophil adhesion to VCAM-1 under both static and flow conditions. Vascular cell adhesion molecule 1 (VCAM-1)2 is a multimodular cell-surface glycoprotein up-regulated on human endothelium in response to pro-inflammatory cytokines in the asthmatic lung (1) and has been implicated in recruitment of eosinophils (EOS) from blood to the airway in asthma (2). Heterodimeric integrin receptors interacting with VCAM-1 are important in the rolling, firm adhesion, and movement of EOS (3, 4). These events are regulated by cytokines, which may alter integrin adhesive activity (5, 6). EOS express at least three potential integrin receptors that may be involved in adhesion to VCAM-1: ␣4␤1 (7-9), ␣D␤2 (7), and ␣4␤7 (8 -11).Considerable information is available about features of human VCAM-1 that are important for adhesion. The loop between ␤ strands C and D in the first immunoglobulin (Ig)-like module is accessible on the protein surface and contains the core integrin-recognition sequence Ile 39 -Asp-Ser-Pro-Leu 43 (IDSPL) (11-15). A second IDSPL site is present in the CD loop in module 4 (12, 14). Based on topology of exons within the mammalian genome and the similarity in amino acid sequence, a three-module unit was likely duplicated to generate VCAM-1 modules 1-3 and 4 -6 (16, 17). Modul...

“…Moreover, treatment with reducing agents, such as dithiothreitol, induced the active conformation of ␤ 1 integrin (33) and increased platelet aggregation through the ␣ IIb ␤ 3 integrin (48). Recently, an anti-␤ 1 antibody with an activationdependent epitope has been mapped to the N-terminal cysteine-rich region, suggesting a role of this region as a regulatory site for integrin activation (33). In addition, several monoclonal antibodies against the C-terminal cysteine-rich regions of ␤ 1 (32,34), ␤ 2 (37), and ␤ 3 (49) integrins have been described as activating mAbs with respect to their ability to promote ligand binding.…”

Section: Discussionmentioning

confidence: 99%

“…The first repeat is less similar to the others and at its N-terminal end contains the cysteine that disulfide bonds to the PSI domain. Several monoclonal antibodies that activate integrins or report conformational changes have been mapped to the C-terminal region of the ␤ subunit that includes the cysteine-rich repeats (28,(32)(33)(34)(35)(36)(37) and to the N-terminal cysteine-rich region (33). Many of these mAbs recognize epitopes that become exposed after integrin activation.…”

Section: From the Center For Blood Research Department Of Pathologymentioning

confidence: 99%

Amino Acid Residues in the PSI Domain and Cysteine-rich Repeats of the Integrin β2 Subunit That Restrain Activation of the Integrin αxβ2

Zang

Springer

2001

The leukocyte integrin ␣ X ␤ 2 (p150,95) recognizes the iC3b complement fragment and functions as the complement receptor type 4. ␣ X ␤ 2 is more resistant to activation than other ␤ 2 integrins and is inactive in transfected cells. However, when human ␣ X is paired with chicken or mouse ␤ 2 , ␣ X ␤ 2 is activated for binding to iC3b. Activating substitutions were mapped to individual residues or groups of residues in the N-terminal plexin/semaphorin/integrin (PSI) domain and C-terminal cysteine-rich repeats 2 and 3. These regions are linked by a long range disulfide bond. Substitutions in the PSI domain synergized with substitutions in the cysteine-rich repeats. Substitutions T4P, T22A, Q525S, and V526L gave full activation. Activation of binding to iC3b correlated with exposure of the CBR LFA-1/2 epitope in cysteine-rich repeat 3. The data suggest that the activating substitutions are present in an interface that restrains the human ␣ X /human ␤ 2 integrin in the inactive state. The opening of this interface is linked to structural rearrangements in other domains that activate ligand binding.The integrin ␣ X ␤ 2 (p150,95, CD11c/CD18) is one of four integrins that are restricted in expression to leukocytes and have different ␣ subunits associated with a common integrin ␤ 2 subunit (1, 2). ␣ X ␤ 2 is also known as the complement receptor type 4. Integrin ␣ X ␤ 2 is expressed on the surface of macrophages, monocytes, granulocytes, and certain activated and B lymphocyte subpopulations (3-6). Upon activation, ␣ X ␤ 2 binds to its ligands, complement component iC3b (7-9) and fibrinogen (5, 10), and mediates leukocyte adherence to endothelium and other cells, possibly by binding to intercellular adhesion molecule 1 (ICAM-1) (4, 11-13).Comparisons among leukocyte integrins suggest that ␣ X ␤ 2 has the highest barrier to activation of ligand binding. On cells that coexpress the integrins ␣ X ␤ 2 and Mac-1 (␣ M ␤ 2 ), stronger cellular activation is required to activate ␣ X ␤ 2 than ␣ M ␤ 2 for binding to the ligand iC3b (8). When transfected into COS or 293T cells, the integrins LFA-1 (␣ L ␤ 2 ) and ␣ M ␤ 2 are active in binding ligands; however, ␣ X ␤ 2 is not (14). Construction of chimeric ␣ M and ␣ X ␣ subunits showed that many reciprocal exchanges activated ligand binding, suggesting that structural perturbations released restraints that otherwise held ␣ X ␤ 2 in an inactive state (14). Interestingly, although ␣ X ␤ 2 expressed on COS-7 cells could not bind to the ligand iC3b, an interspecies hybrid, ␣ X ␤ 2 , comprised of chicken ␤ 2 and human ␣ X subunits was constitutively active in binding iC3b (7). By contrast, human ␣ M /chicken ␤ 2 and human ␣ M /human ␤ 2 integrin heterodimers bound iC3b equally well. Studies with mAb 1 map ligand binding to the I domain of the ␣ X ␤ 2 ␣ X subunit (7). These findings suggest that an intersubunit restraint on ␣ X conformation is loosened with the chicken ␤ 2 subunit so that ␣ X can more readily adopt the conformation that binds iC3b. It may be significant in light of this fi...