Calmodulin regulates L-selectin adhesion molecule expression and function through a protease-dependent mechanism.

Expression of the L-selectin adhesion molecule is rapidly down-regulated upon cell activation through proteolysis at a membrane-proximal site. Here we demonstrate that calmodulin, an intracellular calcium regulatory protein, specifically coprecipitates with L-selectin through a direct association with the cytoplasmic domain of L-selectin. Furthermore, calmodulin inhibitors disrupt L-selectin-dependent adhesion by inducing proteolytic release of L-selectin from the cell surface.

Mutational analysis of the membrane-proximal cleavage site of L-selectin: relaxed sequence specificity surrounding the cleavage site.

L-selectin expression is regulated in part by membrane-proximal cleavage from the cell surface of leukocytes and L-selectin-transfected cells. The downregulation of L-selectin from the surface of neutrophils is speculated to be a process involved in the adhesion cascade leading to neutrophil recruitment to sites of inflammation. We previously reported that L-selectin is cleaved between Lys321 and Ser322 in a region that links the second short consensus repeat (SCR) and the transmembrane domain.

Regulation of L-selectin expression by membrane proximal proteolysis.

L-selectin is a lectin cell adhesion molecule expressed on the cell surfaces of lymphocytes, monocytes and granulocytes. Upon leukocyte activation or L-selectin cross-linking the transmembrane-bound L-selectin is rapidly shed from the cell surface. Based on these observations, it has been proposed that L-selectin is proteolytically cleaved from the cell surface.

Membrane proximal cleavage of L-selectin: identification of the cleavage site and a 6-kD transmembrane peptide fragment of L-selectin.

Rapid downregulation of L-selectin expression occurs in response to leukocyte activation, and it has been speculated to be an integral process in the adhesion cascade leading to neutrophil recruitment to sites of inflammation. It has previously been proposed that L-selectin is proteolytically cleaved from the cell surface; however, the nature of the cleavage site has been unknown. We have produced polyclonal antisera against the extracellular domain and against the cytoplasmic domain of L-selectin.

An assessment of proteolytic enzymes in Tetrahymena thermophila.

Cellular extracts of Tetrahymena thermophila were found to contain substantial levels of proteolytic activity. Protein digestion occurred over broad ranges of pH, ionic strength, and temperature and was stimulated by treatment with thiol reductants, EDTA and sodium dodecyl sulfate. Incubation at temperatures > or = 60 degrees C or with high concentrations of chaotropic reagents such as 10 M urea or 6 M guanidine-HCl caused an apparent irreversible loss of activity.