Binding of the cytoplasmic domain of intercellular adhesion molecule-2 (ICAM-2) to α-actinin

Abstract

Intercellular adhesion molecule-2 (ICAM-2) functions as a ligand for lymphocyte function-associated antigen-1 (LFA-1) and is involved in leukocyte adhesion. We studied intracellular associations of ICAM-2 using a peptide encompassing the cytoplasmic amino acids 231-254 as an affinity matrix. Among the proteins from placental lysates that bound to the peptide was α-actinin as demonstrated by immunoblotting. Purified, 125I-labeled α-actinin also bound to the peptide. Confocal microscopic analysis of Eahy926 cells demonstrated a colocalization of ICAM-2 and α-actinin. Of overlapping octapeptides covering the entire ICAM-2 cytoplasmic amino acids, ICAM- 224241-248 bound α-actinin most avidly and effectively competed with the longer cytoplasmic peptide for binding. The site of interaction in α- actinin was studied using bacterially expressed α-actinin fusion proteins. Several constructs covering nonoverlapping regions of α-actinin bound to the ICAM-2 cytoplasmic peptide suggesting that multiple regions in α-actinin can mediate the interaction. These results, together with previously demonstrated interactions between α-actinin and the adhesion proteins ICAM-1, L-selectin, β1-and β2-integrins emphasize the role of α-actinin as a linker between cell surface adhesion molecules and the actin-containing cytoskeleton.