No abstract

E-selectin, a cytokine-inducible adhesion molecule, supports rolling and stable arrest of leukocytes on activated vascular endothelium. Previous studies have suggested that this transmembrane protein can also transduce signals into the endothelial cell. We now demonstrate activation of the mitogen-activated protein kinase (MAPK) signaling cascade in cultured HUVEC in response to E-selectin-dependent leukocyte adhesion and Ab-mediated cross-linking of cell surface E-selectin. Adhesion of increasing numbers of HL60 cells to IL-1β-activated HUVEC stimulated robust increases in MAPK activity that were abrogated by an E-selectin blocking Ab. Cross-linking of cell surface E-selectin with Abs, as a mimic of multivalent ligand engagement, strongly stimulated MAPK/extracellular signal-related kinase (ERK) kinase (MEK)-dependent MAPK activation and concomitant up-regulation of mRNA for c-fos, an immediate early response gene, whereas Ab cross-linking of HLA class I molecules (present at comparable density) failed to do so. Coimmunoprecipitation documented Ras, Raf-1 and, phospho-MEK complex formation. Unactivated HUVEC transduced with a full-length adenoviral E-selectin construct also exhibited cross-link-induced MAPK activation, macromolecular complex formation, and c-fos up-regulation, whereas HUVEC transduced with a cytoplasmic domain deletion mutant failed to respond. These observations indicate that E-selectin can transduce an activating stimulus via the MAPK cascade into the endothelial cell during leukocyte adhesion.

No abstract

We have demonstrated previously that murine interferon-gamma (MuIFN-gamma) binds to the extracellular domain of the receptor alpha chain through its N-terminus and subsequently to the cytoplasmic domain of the receptor via its C-terminus. Binding of the C-terminus to the cytoplasmic domain of the receptor is thought to occur following endocytosis of the IFN-gamma-receptor complex. In fact, the MuIFN-gamma C-terminus peptide, MuIFN-gamma (95-133), has full agonist activity on macrophages where it is internalized through pinocytosis. Here we examine the structural elements required for the agonist activity of MuIFN-gamma (95-133). Disruption of the alpha helical structure of the peptide by proline substitutions or truncation of the helix resulted in significant loss of binding or loss of antiviral activity or both and induction of MHC class II molecules. Further, removal of the polycationic sequence RKRKR in the tail beyond the helical structure also resulted in loss of agonist activity. Thus, we have isolated the functional site on MuIFN-gamma to the C-terminus and have shown that its helical structure and polycationic tail are required for binding to the cytoplasmic domain of the receptor and induction of biologic activity.

No abstract