Abstract
The beta(2) integrin lymphocyte function-associated antigen-1 (LFA-1) is a conformationally flexible alpha/beta heterodimeric receptor, which is expressed on the surface of all leukocytes. LFA-1 mediates cell adhesion crucial for normal immune and inflammatory responses. Intracellular signals or cations are required to convert LFA-1 from a nonligand binding to a ligand binding state. Here we investigated the effect of small molecule inhibitors on LFA-1 by monitoring the binding of monoclonal antibodies mapped to different receptor domains. The inhibitors were found to not only induce epitope changes in the I domain of the alpha(L) chain but also in the I-like domain of the beta(2) chain depending on the individual chemical structure of the inhibitor and its binding site. For the first time, we provide strong evidence that the I-like domain represents a target for allosteric LFA-1 inhibition similar to the well established regulatory L-site on the I domain of LFA-1. Moreover, the antibody binding patterns observed in the presence of the various inhibitors establish a conformational interaction between the LFA-1 I domain and the I-like domain in the native receptor that is formed upon activation. Differentially targeting the binding sites of the inhibitors, the L-site and the I-like domain, may open new avenues for highly specific therapeutic intervention in diseases where integrins play a pathophysiological role.
Highlights
Integrins are a family of ␣/ heterodimeric cell surface receptors that mediate cell-cell and cell-extracellular matrix interactions
This result is in agreement with previous studies showing that lymphocyte function-associated antigen-1 (LFA-1) binds to ICAM-1 and ICAM-2 stronger than to ICAM-3 [26]
The IC50 values determined for the LFA-1 inhibitors in the HUT78/ICAM-1 adhesion assay were found to be similar to IC50 values determined in an ICAM-1 adhesion assay utilizing the human lymphoma cell line Jurkat
Summary
Transfectants expressing mutant LFA-1 with alanine or tryptophan substitutions in the C-terminal helix region of the I domain show impaired or constitutively active binding to ICAM-1 [19, 20] The importance of this region in controlling LFA-1 activity is further underlined by the fact that small molecule LFA-1 inhibitors bind to a hydrophobic pocket between the C-terminal helix and the central -sheet of the LFA-1 I domain, termed the. Utilizing the native LFA-1 receptor, as compared with mutated LFA-1 studied previously [14], we provide strong evidence for a functional relationship between the I domain and the I-like domain, which is induced upon activation
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