title: Identification of the binding site in intercellular adhesion molecule 1 for its receptor, leukocyte function-associated antigen 1.
abstract: Intercellular adhesion molecule 1 (ICAM-1, CD54) is a member of the Ig superfamily and is a counterreceptor for the beta 2 integrins: lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18), complement receptor 1 (MAC-1, CD11b/CD18), and p150,95 (CD11c/CD18). Binding of ICAM-1 to these receptors mediates leukocyte-adhesive functions in immune and inflammatory responses. In this report, we describe a cell-free assay using purified recombinant extracellular domains of LFA-1 and a dimeric immunoadhesin of ICAM-1. The binding of recombinant secreted LFA-1 to ICAM-1 is divalent cation dependent (Mg2+ and Mn2+ promote binding) and sensitive to inhibition by antibodies that block LFA-1-mediated cell adhesion, indicating that its conformation mimics that of LFA-1 on activated lymphocytes. We describe six novel anti-ICAM-1 monoclonal antibodies, two of which are function blocking. Thirty-five point mutants of the ICAM-1 immunoadhesin were generated and residues important for binding of monoclonal antibodies and purified LFA-1 were identified. Nineteen of these mutants bind recombinant LFA-1 equivalently to wild type. Sixteen mutants show a 66-2500-fold decrease in LFA-1 binding yet, with few exceptions, retain binding to the monoclonal antibodies. These mutants, along with modeling studies, define the LFA-1 binding site on ICAM-1 as residues E34, K39, M64, Y66, N68, and Q73, that are predicted to lie on the CDFG beta-sheet of the Ig fold. The mutant G32A also abrogates binding to LFA-1 while retaining binding to all of the antibodies, possibly indicating a direct interaction of this residue with LFA-1. These data have allowed the generation of a highly refined model of the LFA-1 binding site of ICAM-1.
authors.affiliation: Department of Immunology, Genentech Inc., South San Francisco, California 94080, USA.
authors.forename: K L
authors.initials: KL
authors.lastname: Fisher
authors.forename: J
authors.initials: J
authors.lastname: Lu
authors.forename: L
authors.initials: L
authors.lastname: Riddle
authors.forename: K J
authors.initials: KJ
authors.lastname: Kim
authors.forename: L G
authors.initials: LG
authors.lastname: Presta
authors.forename: S C
authors.initials: SC
authors.lastname: Bodary
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D001666:Binding Sites, Antibody; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D016169:Lymphocyte Function-Associated Antigen-1 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D019010:Vascular Cell Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D016169:Lymphocyte Function-Associated Antigen-1; D011994:Recombinant Proteins; D019010:Vascular Cell Adhesion Molecule-1; D018799:Intercellular Adhesion Molecule-1