Homophilic adhesion of human CEACAM1 involves N-terminal domain interactions: structural analysis of the binding site

CEACAM1 on leukocytic, endothelial, and epithelial cells functions in homop hilic adhesion, tumor suppression, regulating cell adhesion and proliferati on, and in heterophilic adhesion as a receptor for E-selectin and Neisseria meningiditis, Neisseria gonorrhoeae, Haemophilus influenzae, and murine co ronaviruses. The 8 transmembrane isoforms of human CEACAM1 possess an extra cellular N-terminal IgV domain, followed by variable numbers of IgC2 domain s. To establish which key amino acids contribute specifically to CEACAM1 ho mophilic adhesion, exposed amino acids in the N-terminal domain of a solubl e form of CEACAM1 were subjected to mutagenesis. Analyses of mutant protein s with conformationally dependent antibodies indicated that most mutations did not substantially affect the structural integrity of CEACAM1. Neverthel ess, decreased adhesion was observed for the single mutants V39A or D40A (s ingle-letter amino acid codes) in the CC ' loop and for the triple mutants located in the GFCC 'C " face of the N-terminal domain. Interestingly, wher eas single mutations in R64 or D82 that are predicted to form a salt bridge between the base of the D and F beta strands close to the critical V39 and D40 residues also abolish adhesion, an amino acid swap (R64D and D82R), wh ich maintains the salt bridge was without significant effect. These studies indicate that the CC ' loop plays a crucial role in the homophilic adhesio n of CEACAM1. They further predict that specific hydrophobic amino acid res idues on the nonglycosylated GFCC 'C " face of CEACAM1 N-terminal domain ar e not only involved in heterophilic interactions with Opa proteins and H in fluenzae, but are also critical for protein-protein interactions between 2 CEACAM1 molecules on opposing cells. (C) 2001 by The American Society of He matology.