α4 Integrin Binding Interfaces on VCAM-1 and MAdCAM-1

Integrins ␣ E ␤ 7 and ␣ 4 ␤ 7 are involved in localization of leukocytes at mucosal sites. Although both ␣ E ␤ 7 and ␣ 4 ␤ 7 utilize the ␤ 7 chain, they have distinct binding specificities for E-cadherin and mucosal addressin cell adhesion molecule-1 (MAdCAM-1), respectively. We found that mutation of the metal ion-dependent adhesion site (MIDAS) in the ␣ E A-domain (D190A) abolished E-cadherin binding, as did mutation F298A on the A-domain surface near the MIDAS cleft. A docking model of the A-domain with E-cadherin domain 1 indicates that coordination of the ␣ E MIDAS metal ion by E-cadherin Glu 31 and a novel projection of Phe 298 into a hydrophobic pocket on E-cadherin provide the basis for the interaction. The location of the binding site on the ␣ E A-domain resembles that on other integrins, but its structure appears distinctive and particularly adapted to recognize the tip of E-cadherin, a unique integrin ligand. Additionally, mutation of the ␤ 7 MIDAS motif (D140A) abolished ␣ E ␤ 7 binding to E-cadherin and ␣ 4 ␤ 7 -mediated adhesion to MAdCAM-1, and ␣ 4 chain mutations that abrogated binding of ␣ 4 ␤ 1 to vascular cell adhesion molecule-1 and fibronectin similarly reduced ␣ 4 ␤ 7 interaction with MAdCAM-1. Thus, although specificity can be determined by the integrin ␣ or ␤ chain, common structural features of both subunits are required for recognition of dissimilar ligands.Integrins are heterodimeric glycoproteins consisting of noncovalently associated ␣ and ␤ subunits that play diverse roles in cell-cell and cell-matrix interactions. Integrins of the ␤ 1 , ␤ 2 , and ␤ 7 subfamilies are critical for leukocyte homing (1, 2). For example, binding of integrin ␣ 4 ␤ 7 to mucosal addressin cell adhesion molecule-1 (MAdCAM-1) 1 on intestinal endothelial cells is required for the homing of naive lymphocytes to Peyer's patches (2). Integrins are also thought to play a role in microenvironmental homing or retention of lymphocytes at particular sites within a tissue (2). For instance, integrin ␣ E ␤ 7 binds to epithelial cadherin (E-cadherin) and is involved in retention of lymphocytes within the epithelium (3-6).Many integrin ligands are members of the immunoglobulin superfamily (such as VCAM-1 and MAdCAM-1) or possess domains that resemble the immunoglobulin fold (e.g. E-cadherin and the type III repeats of fibronectin). A unifying feature of these ligands is an integrin-binding surface containing an exposed acidic amino acid. For example, ␣ 4 ␤ 1 and ␣ 4 ␤ 7 bind to VCAM-1 and MAdCAM-1, respectively, on the face of domain 1 formed by the C, F, and G ␤-strands, centered on an aspartate residue in the loop connecting the C and D strands (7-9). Domain 1 of E-cadherin contains a glutamate residue at the tip of the BC-loop essential for ␣ E ␤ 7 binding (10,11), and the tenth type III repeat of fibronectin has an acidic residue within an RGD sequence on the FG-loop required for ␣ 5 ␤ 1 binding (12). Other integrin ligands such as collagen I and iC3b that are not known to adopt immunoglobulin-like folds also possess...

Section: Discussionmentioning

confidence: 99%

The Role of α and β Chains in Ligand Recognition by β7 Integrins

Higgins¹,

Cernadas²,

Tan³

et al. 2000

“…Each of these proteins contains putative integrin binding sequences in the linker between ␤ strands C and D that is at the position of the IDSPL sequence in modules 1 and 4 of human VCAM-1 (55). Integrins ␣M␤2 (56) and ␣L␤2 (57) contain an I domain in the ␣ subunit.…”

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...

“…Although previous studies have revealed that some residues in MAdCAM-1 are important for MAdCAM-1-␣ 4 ␤ 7 binding (9,12,13,15), the structural basis for supporting MAdCAM-1-␣ 4 ␤ 7 -mediated rolling and firm cell adhesion remains elusive because the static cell adhesion assay used in those studies is unable to distinguish rolling and firm cell adhesion. In this study, we used a flow chamber assay to screen the critical residues in MAdCAM-1, which are important for supporting rolling and firm cell adhesions, respectively.…”

Section: Discussionmentioning

confidence: 93%

“…The essential integrin-binding motif (LDTS) resides in the CCЈ loop of MAdCAM-1 D1, and the Asp-42 in the LDTS motif serves as the primary ␣ 4 ␤ 7 -binding site by directly interacting with the metal ion at the metal ion-dependent adhesion site (MIDAS) in the integrin ␤ 7 I domain (8,(12)(13)(14). Mutational studies suggest that some other residues in the CCЈ loop of MAdCAM-1 are also involved in MAdCAM-1-␣ 4 ␤ 7 binding (9,12,13). The DE loop in D2 is predominated by negatively charged residues, which have been reported to play an important role in determining integrin binding specificity (15).…”

mentioning

confidence: 99%

“…The crystal structure of MAdCAM-1 highlights two protruding loops from the two Ig domains, the CCЈ loop in D1 and DE loop in D2, which are important for the interaction between MAdCAM-1 and ␣ 4 ␤ 7 (10,11). The essential integrin-binding motif (LDTS) resides in the CCЈ loop of MAdCAM-1 D1, and the Asp-42 in the LDTS motif serves as the primary ␣ 4 ␤ 7 -binding site by directly interacting with the metal ion at the metal ion-dependent adhesion site (MIDAS) in the integrin ␤ 7 I domain (8,(12)(13)(14). Mutational studies suggest that some other residues in the CCЈ loop of MAdCAM-1 are also involved in MAdCAM-1-␣ 4 ␤ 7 binding (9,12,13).…”

mentioning

confidence: 99%

See 1 more Smart Citation

The CC′ and DE Loops in Ig Domains 1 and 2 of MAdCAM-1 Play Different Roles in MAdCAM-1 Binding to Low- and High-affinity Integrin α4β7

Sun¹,

Wu²,

Qi³

et al. 2011

Lymphocyte homing is regulated by the dynamic interaction between integrins and their ligands. Integrin ␣ 4 ␤ 7 mediates both rolling and firm adhesion of lymphocytes by modulating its affinity to the ligand, mucosal addressin cell adhesion molecule-1 (MAdCAM-1). Although previous studies have revealed some mechanisms of ␣ 4 ␤ 7 -MAdCAM-1 binding, little is known about the different molecular bases of the low-and high-affinity ␣ 4 ␤ 7 -MAdCAM-1 interactions, which mediate rolling and firm adhesion of lymphocytes, respectively. Here, we found that two loops in immunoglobulin domains 1 and 2 (D1 and D2) of MAdCAM-1 played different roles in MAdCAM-1 binding to low-affinity (inactive) and high-affinity (activated) ␣ 4 ␤ 7 . The Asp-42 in the CC loop of D1 was indispensable for MAdCAM-1 binding to both low-affinity and high-affinity ␣ 4 ␤ 7 . The other CC loop residues except for Arg-39 and Ser-44 were essential for MAdCAM-1 binding to both inactive ␣ 4 ␤ 7 and ␣ 4 ␤ 7 activated by SDF-1␣ or talin, but not required for MAdCAM-1 binding to Mn 2؉ -activated ␣ 4 ␤ 7 . Single amino acid substitution of the DE loop residues mildly decreased MAdCAM-1 binding to both inactive and activated ␣ 4 ␤ 7 . Notably, removal of the DE loop greatly impaired MAdCAM-1 binding to inactive and SDF-1␣-or talin-activated ␣ 4 ␤ 7 , but only decreased 60% of MAdCAM-1 binding to Mn 2؉ -activated ␣ 4 ␤ 7 . Moreover, DE loop residues were important for stabilizing the low-affinity ␣ 4 ␤ 7 -MAdCAM-1 interaction. Thus, our findings demonstrate the distinct roles of the CC and DE loops in the recognition of MAdCAM-1 by low-and high-affinity ␣ 4 ␤ 7 and suggest that the inactive ␣ 4 ␤ 7 and ␣ 4 ␤ 7 activated by different stimuli have distinct conformations with different structural requirements for MAdCAM-1 binding.Lymphocyte homing from circulation to lymphoid tissues and sites of inflammation is regulated by the dynamic interaction between lymphocyte integrin receptors and endothelial immunoglobulin superfamily cell adhesion molecules (1-3).Integrin ␣ 4 ␤ 7 is an important lymphocyte homing receptor that can mediate both rolling and firm adhesion of lymphocytes, two of the critical steps in lymphocyte migration and tissue-specific homing (4). Its ligand, MAdCAM-1, 2 is preferentially expressed on high endothelial venules of gut-associated lymphoid organs and on lamina propria venules, helping lymphocyte traffic to mucosal organs (5). The interaction between MAdCAM-1 and integrin ␣ 4 ␤ 7 is the key step in lymphocyte homing to gut and plays vital roles in both gut mucosal immune homeostasis and intestinal inflammation (6, 7).Human MAdCAM-1 is a multidomain molecule consisting of two Ig-like domains followed by mucin-like sequences (8). Domain swapping experiments with MAdCAM-1 and VCAM-1 have demonstrated the requirement of the two Ig domains of MAdCAM-1 for efficient integrin ␣ 4 ␤ 7 binding (9). The crystal structure of MAdCAM-1 highlights two protruding loops from the two Ig domains, the CCЈ loop in D1 and DE loop in D2, which are important fo...