title: Regulation of L-selectin expression by membrane proximal proteolysis.
abstract: 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. However a panel of common protease inhibitors have no effect on L-selectin proteolysis. To further define the mechanism of L-selectin down-regulation we have produced reagents to study proteolytic fragments of L-selectin. We have developed a trapping ELISA for the detection of soluble L-selectin. In addition we have produced a high affinity polyclonal antisera against the extracellular domain and against the cytoplasmic domain of L-selectin. Both antisera immunoprecipitate the intact form of L-selectin from metabolically labeled PHA lymphoblasts and peripheral blood neutrophils. We review here our progress in defining a 6 kD L-selectin transmembrane peptide (L-STMP) from PMA activated lymphoblasts and fMLP-activated neutrophils. Radiochemical sequencing data indicate that the cleavage site occurs between Lys321 and Ser322 in a short membrane-proximal region of the extracellular domain.
authors.affiliation: Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT 06877, USA.
authors.forename: T K
authors.initials: TK
authors.lastname: Kishimoto
authors.forename: J
authors.initials: J
authors.lastname: Kahn
authors.forename: G
authors.initials: G
authors.lastname: Migaki
authors.forename: E
authors.initials: E
authors.lastname: Mainolfi
authors.forename: F
authors.initials: F
authors.lastname: Shirley
authors.forename: R
authors.initials: R
authors.lastname: Ingraham
authors.forename: R
authors.initials: R
authors.lastname: Rothlein
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D015815:Cell Adhesion Molecules / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology*; D010450:Endopeptidases / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007163:Immunosorbent Techniques; D019041:L-Selectin; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000378:metabolism; D008969:Molecular Sequence Data; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D017385:Sequence Homology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D015815:Cell Adhesion Molecules; D010446:Peptide Fragments; D011480:Protease Inhibitors; D019041:L-Selectin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D010450:Endopeptidases; D013755:Tetradecanoylphorbol Acetate