E-Selectin (ELAM-1, CD62E), a cytokine-inducible, endothelial-specific member of the Selectin family of adhesion receptors, supports the rolling and stable arrest of selected blood leukocytes on the surface of activated vascular endothelium in vitro and in vivo. Recent data indicate that, in addition to this adhesion-supporting function, E-selectin also is functioning in the transduction of signals into the endothelial cells, and in biomechanical events such as cytoskeletal anchoring. In this continuing project, we will test the hypothesis that the E-selectin molecule mediates intercellular and intracellular communication during leukocyte-endothelial adhesive interactions at sites of inflammation. In Specific Aim #1, the kinetics and topography of formation, and biochemical composition, of "apical focal adhesion complexes (apical FACs)", induced by E-selectin-dependent adhesion of leukocytes to activated endothelial cells, will be characterized using a combination of confocal immunofluorescence microscopy, immuno-electron microscopy and immunochemistry. In Specific Aim #2, the mechanisms that regulate the phosphorylation/ dephosphorylation of the cytoplasmic domain of E-selectin under various conditions of leukocyte-endothelial interaction will be examined; the involvement of specific amino acid residues will be determined by mutagenesis; and the functional significance for cytoskeletal association, apical FAC formation and intracellular signaling events will be explored. In Specific Aim #3, a "downstream" molecular targets of E-selectin- mediated signaling in the endothelial cell will be investigated, including: mitogen-activate protein kinase (MAP-kinase) activation; lateral endothelial-expressed adhesion molecules and cytokines relevant to the inflammatory response; and autoregulation of endothelial E-selectin expression by E-selectin-transduced signals. In Specific AIM #4, the pathophysiologic consequences of E-selectin- dependent intercellular and intracellular signaling will be examined in various in vitro and in vivo models of leukocyte-endothelial interaction, making use of unique cellular and molecular biological reagents developed in this project, including E-selectin-deficient mice, cultured microvascular endothelial cells derived from these animals, and adenoviral vectors for the efficient and tiratable expression of wildtype and mutant E-selectin molecules. The results of these studies should increase our understanding of the active role of vascular endothelium in response-to-injury and inflammatory reactions and, in particular, the potential contribute of E-selecting to intercellular and intracellular communication during thee processes.