IDENTIFICATION OF AMINO-ACIDS IN THE CD11A I-DOMAIN IMPORTANT FOR BINDING OF THE LEUKOCYTE FUNCTION-ASSOCIATED ANTIGEN-1 (LFA-1) TO INTERCELLULAR-ADHESION MOLECULE-1 (ICAM-1)

Leukocyte function-associated antigen-1 (LFA-1) is a cell surface adhe sion receptor for intercellular adhesion molecule-1, -2, and -3 (ICAM- 1, -2, -3). Using human/murine chimeras of the I domain of the LFA-1 a lpha subunit (CD11a), we recently identified the epitopes recognized b y eight monoclonal antibodies against CD11a that inhibit LFA-1 binding to ICAM-1. In this report, we determined that replacement of the enti re human I domain with the entire murine I-domain in CD11a completely abrogated LFA-1 binding to human ICAM-1 without affecting the gross co nformation or heterodimer formation of LFA-1, as assayed by antibody b inding, In order to assess which residues of the I-domain are responsi ble for binding to ICAM-1, we tested the ability of a panel of human/m urine I-domain chimeras to bind to human ICAM-1. When complexed with C D18, all CD11a chimeras bound ICAM-1 at levels comparable to wild-type CD11a/CD18, indicating that the residues in these chimeras which diff er in human and murine I domains may not play a critical role in LFA-1 binding to ICAM-1. A series of point mutations of residues that are c onserved between murine and human CD11a I-domains, as well as between CD11b and CD11c, were also generated, Substitution of alanine for prol ine at position 192 in the human CD11a I-domain abrogated adhesion of LFA-1 to ICAM-1. Antibody binding data suggested that this was due to conformational changes within the I domain. Mutation of the aspartic a cids at positions 137 and 239 to either alanine or lysine completely d estroyed ICAM-1 binding. The conformation of LFA-1 alanine mutants was not significantly altered. This suggests that these aspartic acids ar e required for binding of human LFA-1 to human ICAM-1.