Evidence of a functional role for interaction between ICAM-1 and nonmuscle alpha-actinins in leukocyte diapedesis.
ICAM-1 is involved in both adhesion and extravasation of leukocytes to endothelium during inflammation. It has been shown that the ICAM-1 cytoplasmic domain is important for transendothelial migration of leukocytes but the precise molecular mechanisms involving the intracytoplasmic portion of ICAM-1 is not known. To characterize precisely the molecular scaffolding associated with ICAM-1, we have used the yeast two-hybrid system, and we have identified six different proteins interacting with the ICAM-1 cytoplasmic domain. In this study, we report that the two forms of nonmuscle alpha-actinin (i.e., alpha-actinin 1 and alpha-actinin 4) associate with ICAM-1, and that these interactions are essential for leukocyte extravasation. These interactions were further confirmed by coimmunoprecipitation and immunofluorescence in endothelial cells and in ICAM-1-transfected Chinese hamster ovary cells. The function of these interactions was analyzed by point mutation of charged amino acids located on ICAM-1 cytoplasmic domain. We have identified three charged amino acids (arginine 480, lysine 481, and arginine 486) which are essential in the binding of alpha-actinins to the ICAM-1 cytoplasmic tail. Mutation of these amino acids completely inhibited ICAM-1-mediated diapedesis. Experiments with siRNA inhibiting specifically alpha-actinin 1 or alpha-actinin 4 on endothelial cells indicated that alpha-actinin 4 had a major role in this phenomenon. Thus, our data demonstrate that ICAM-1 directly interacts with cytoplasmic alpha-actinin 1 and 4 and that this interaction is required for leukocyte extravasation.
Celli
Lionel
L
Institut National de la Santé et de la Recherche Médicale, Unité 578, Grenoble, France, and Université Grenoble I, Groupe de Recherche sur le Cancer du Poumon, Institut Albert Bonniot, Grenoble, France.
Ryckewaert
Jean-Jacques
JJ
Delachanal
Elisabeth
E
Duperray
Alain
A
D000185:Actinin / Q000378:metabolism / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002465:Cell Movement / Q000276:immunology*; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D042783:Endothelial Cells / Q000166:cytology / Q000502:physiology*; D006801:Humans; D007249:Inflammation / Q000378:metabolism / Q000473:pathology; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D007962:Leukocytes / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D008840:Microfilament Proteins / Q000502:physiology*; D008969:Molecular Sequence Data
C111453:ACTN4 protein, human; D008840:Microfilament Proteins; D000185:Actinin; D018799:Intercellular Adhesion Molecule-1