The I Domain of Integrin Leukocyte Function-associated Antigen-1 Is Involved in a Conformational Change Leading to High Affinity Binding to Ligand Intercellular Adhesion Molecule 1 (ICAM-1)

Alison Mcdowall,Birgit Leitinger,Paula Stanley,Paul A Bates,Anna M Randi,Nancy Hogg

doi:10.1074/jbc.273.42.27396

Abstract

On T cells the leukocyte integrin leukocyte function-associated antigen-1 (LFA-1) (CD11a/CD18) can be induced to bind its ligand intercellular adhesion molecule 1 (ICAM-1) (CD54) either by increasing the affinity of the receptor with Mg2+ and EGTA or by receptor clustering following activation with phorbol ester. The existence of these two adhesion-inducing pathways implies that alternative mechanisms might exist by which LFA-1 engages ICAM-1. The LFA-1 alpha subunit I domain contains a major binding site for ICAM-1. In this study we show that soluble LFA-1 I domain blocks ICAM-1 binding of the high affinity Mg2+-induced form of LFA-1 but not the phorbol ester-induced form. Under conditions of Mg2+-activation, the soluble I domain also prevents expression of an activation dependent epitope on LFA-1, implying that it inhibits a conformational change necessary for conversion to the high affinity form of this integrin. In addition, the binding of Mg2+-activated LFA-1 to ICAM-1 is blocked by peptides covering the alpha4-beta3 loop, the beta3-alpha5 loop, and the alpha5 helix of the I domain, whereas none of the peptides tested blocks phorbol ester-mediated adhesion. The blocking peptides localize to the same face of the crystal structure of the LFA-1 I domain and define an area that, during activation, may be involved in association of the I domain with another region of LFA-1, potentially the beta-propeller domain. This is the first evidence linking a structural domain of an integrin, in this case the I domain, with a particular activation mechanism.

Highlights

Adhesion mediated by the interaction of the integrin LFA-11 (CD11a/CD18) with one of its ligands, intercellular adhesion molecule 1 (ICAM-1), ICAM-2, or ICAM-3, is crucial to the inflammatory process
leukocyte functionassociated antigen-1 (LFA-1) I Domain Blocks Mg2ϩ but Not Phorbol Ester-induced Adhesion—The role of the I domain in different forms of LFA1-mediated adhesion to ICAM-1 was investigated by testing the ability of soluble recombinant (Leu111–Ser327)I dom to interfere with Mg2ϩ/EGTA or PdBu-stimulated T cell binding to immobilized ICAM-1
One possible reason for the failure of the (Leu111–Ser327)I dom to block PdBu-mediated adhesion is that PdBu might induce a higher overall binding strength than is achieved with Mg2ϩ/EGTA and the binding of Mg2ϩ/EGTA-treated cells would be more inhibited than the binding of cells treated with phorbol ester

Summary

EXPERIMENTAL PROCEDURES

The LFA-1 I domain residues Leu111–Ser327, termed (Leu111–Ser327)I dom, includes the predicted I domain sequence and an amino-terminal extension of ϳ17 amino acids [30]. Plates that had been ICAM-1Fc-coated and bovine serum albumin-blocked were incubated for 30 min at 37 °C with 50 ␮l of proteins or mAbs in HEPES buffer containing 2 mM MgCl2, 2 mM EGTA. The solutions were removed, and 50 ␮l of HEPES buffer containing 2 mM MgCl2, 2 mM EGTA were added followed by the addition of 50 ␮l of BCECF/AM-labeled cells (2 ϫ 105/well). BCECF/AM-labeled cells (2– 4 ϫ 106/ml) in HEPES buffer containing 1 mM MgCl2, 1 mM EGTA were incubated in the presence or absence of proteins or mAbs for 15 min at 37 °C. After centrifugation for 1 min at 30 ϫ g, the solutions were removed, and the cells were resuspended in HEPES buffer and added to ICAM-1Fccoated, bovine serum albumin-blocked, and washed wells containing 50 ␮l of HEPES buffer and 2 mM MgCl2, 2 mM EGTA. In experiments testing the effect of peptides on mAb 24 expression, peptides were added instead of the soluble I domain, and the same procedure was followed

Preparation and Characterization of Synthetic Peptides

RESULTS

Amino acid sequence

DISCUSSION