ICAM1 ligation‐induced transmigratory complex formation regulates leukocyte transendothelial migration

Abstract

ICAM1 expression is upregulated on activated endothelium and is important in leukocyte adhesion and transendothelial migration (TEM). ICAM1 engagement initiates several signaling events including cortactin phosphorylation and association with the ICAM1 cytoplasmic tail. We hypothesized that interactions between cortactin and a putative SH3 binding domain in the ICAM1 tail regulate leukocyte TEM. To test this we mutated 3 amino acids within the ICAM1 cytoplasmic tail such that the putative SH3 binding domain was no longer present (SH3Mut ICAM1). We used adenoviral infection to express WT ICAM1 or SH3Mut ICAM1 in human umbilical vein endothelial cells (HUVEC). Standard biochemical methods and transmigration assays were used to assess functional relevance of the SH3 mutation. ICAM1 engagement triggers cortactin association with ICAM1. Approximately 50% less cortactin associates with SH3Mut ICAM1 (n=3, p<0.05). Neutrophil TEM is also reduced by 43% in cells expressing SH3Mut ICAM1. Furthermore, ICAM1 ligation induces the formation of a large transmigratory complex which includes ICAM1, VE‐Cad, p120, pSrc, and cortactin. Formation of this complex is, in part, dependent on the SH3 binding domain. Our study identifies an SH3 binding motif in the ICAM‐1 cytoplasmic tail that is necessary for association with cortactin and provides evidence that transmigratory complex formation is important for TEM.