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 adhesion 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 alpha subunit (CD11a), we recently identified the epitopes recognized by eight monoclonal antibodies against CD11a that inhibit LFA-1 binding to ICAM-1. In this report, we determined that replacement of the entire human I-domain with the entire murine I-domain in CD11a completely abrogated LFA-1 binding to human ICAM-1 without affecting the gross conformation or heterodimer formation of LFA-1, as assayed by antibody binding. In order to assess which residues of the I-domain are responsible for binding to ICAM-1, we tested the ability of a panel of human/murine I-domain chimeras to bind to human ICAM-1. When complexed with CD18, all CD11a chimeras bound ICAM-1 at levels comparable to wild-type CD11a/CD18, indicating that the residues in these chimeras which differ 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 conserved between murine and human CD11a I-domains, as well as between CD11b and CD11c, were also generated. Substitution of alanine for proline 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 acids at positions 137 and 239 to either alanine or lysine completely destroyed ICAM-1 binding. The conformation of LFA-1 alanine mutants was not significantly altered. This suggests that these aspartic acids are required for binding of human LFA-1 to human ICAM-1.
Edwards
C P
CP
Department of Immunology, Genentech, Inc., South San Francisco, California 94080, USA.
Champe
M
M
Gonzalez
T
T
Wessinger
M E
ME
Spencer
S A
SA
Presta
L G
LG
Berman
P W
PW
Bodary
S C
SC
D000818:Animals; D002448:Cell Adhesion / Q000235:genetics / Q000502:physiology; D002712:Chlorides / Q000494:pharmacology; D004252:DNA Mutational Analysis; D000939:Epitopes; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism*; D008024:Ligands; D016169:Lymphocyte Function-Associated Antigen-1 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015636:Magnesium Chloride / Q000494:pharmacology; D017895:Manganese Compounds / Q000494:pharmacology; D051379:Mice; D017354:Point Mutation; D011485:Protein Binding / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013045:Species Specificity; D013329:Structure-Activity Relationship
D002712:Chlorides; D000939:Epitopes; D008024:Ligands; D016169:Lymphocyte Function-Associated Antigen-1; D017895:Manganese Compounds; D011993:Recombinant Fusion Proteins; D015636:Magnesium Chloride; D018799:Intercellular Adhesion Molecule-1; C025340:manganese chloride