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

Abstract

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...

Role of the αI domain in ligand binding by integrin αEβ7

“…A report that mutations in the MIDAS of the g 7 subunit in § E g 7 block recognition of E-cadherin [28] is consistent with this view but is also compatible with an alternative interpretation that the g subunit is directly involved in ligand binding. The present observation that blocking antibodies to the g I domain inhibit function of the wild-type § I domain, but not the locked-open form, supports the first interpretation and agrees with earlier findings on LFA-1 [19] More direct evidence that the § E I domain provides the sole contact site for E-cadherin comes from our experiments comparing cadherin monomers and dimers.…”

“…Two additional residues were mutated: F319 (purple) which is predicted to undergo a major increase in solvent accessibility in the open conformation [3] and D317 (green) which undergoes minor displacement. D317 was chosen because mutations to alanine in the same position in human § E (E325A) [28] or to lysine in § M (D273K) [3] were reported to enhance adhesion. Fig.…”

“…Antibodies specific for the I domain of the g 7 subunit ablate the interaction between § 4 g 7 and MAdCAM-1, VCAM-1 or fibronectin [32] and mutations in the g 7 MIDAS had a similar effect on adhesion of § E g 7 to Ecadherin [28]. Fig.…”

“…The only characterized ligand for this integrin is E-cadherin, which is engaged by § E g 7 through a binding site on the distal surface of the N-terminal domain (EC1) of the cadherin molecule. A persuasive model for § E I domain docking to this site has been proposed [28]. E-cadherin is thought to be expressed on the cell surface as a cis-dimer formed by contacts between adjacent EC1 domains [29] and recent studies have suggested that dimerization may be required for recognition by § E g 7 [30].…”

Integrin αE(CD103)β7 Mediates Adhesion to Intestinal Microvascular Endothelial Cell Lines Via an E-Cadherin-Independent Interaction

“…Clones producing the greatest quantity of soluble heterodimer were identified based on sandwich ELISA of the supernatant (43) using the anti-␤ 7 mAb Fib504 for capture and the biotinylated anti-␣ E mAb ␣E7-1 for detection. For biochemical analysis, 20 ml of supernatant was passed over a 300-l protein G column, and the adsorbed protein was analyzed by SDS-PAGE followed by Coomassie blue staining as described previously (predicted M r : ␣␤-Fc ϭ 280 kDa, ␣-Fc ϭ 160 kDa, ␤-Fc ϭ 120 kDa, ␣␣-Fc ϭ 320 kDa, ␤␤-Fc ϭ 240 kDa) (44). A soluble ␣ E construct containing a mutation in the metal ion-dependent adhesion site (MIDAS) was generated by exchanging fragment 236-1256 of the ␣ E -Fc with a similar fragment from the full-length ␣ E (D190A) subunit (44) using BpuII02I sites.…”