Leukocyte recruitment is an essential component of the pathogenesis of inflammatory disorders. The regulation of leukocyte recruitment into sites of inflammation occurs principally at the level of leukocyte recognition of endothelium, a process which is initiated by a family of molecules designated selectins. This proposal is directed at understanding the molecular and cellular basis of adhesion mediated by L-selectin, the only selectin expressed on leukocytes. This adhesion molecule is expressed on all classes of leukocytes, which have distinct patterns of migration in vivo, and appears to be involved in many types of leukocyte recruitment during inflammation. In spite of intense investigation, the basis for adhesion mediated by L-selectin is only partially understood. Our preliminary studies indicate that interactions between L-selectin and the actin cytoskeleton via a-actinin and vinculin are essential for L-selectin mediated adhesion.
In specific aim 1, the molecular basis and functional significance of L-selectin/a-actinin interactions will be studied. Immunoprecipitation and Western blotting from cell lines transfected with various L-selectin cytoplasmic tail mutants will be used to identify the residues of the L-selectin cytoplasmic tail responsible for binding to a-actinin and vinculin. The functional significance of these interactions will be determined by examination of the ability of this panel of transfected cell lines to bind to high endothelial venules (HEV) of lymph nodes and to roll in vivo, the two best characterized and most important functions mediated by L-selectin.
In specific aim 2, other proteins which interact with the L-selectin cytoplasmic tail will be identified, and cDNA encoding these proteins will be cloned.
In specific aim 3, immunoelectron microscopy will be used to explore the molecular basis and functional importance of L-selectin positioning into the microvilli of leukocytes, a location believed to be important to the function of L-selectin, with particular attention to the cytoskeletal proteins involved in this protein sorting phenomenon. These studies should add considerably to our understanding of how L-selectin works, and offer the possibility of multiple new clinical targets for intervention in many acute and chronic inflammatory disorders.
