title: Protease-resistant L-selectin mutants. Down-modulation by cross-linking but not cellular activation.
abstract: The adhesion molecule L-selectin (CD62L) is rapidly shed from the plasma membrane during leukocyte activation as a result of proteolytic cleavage between Lys321 and Ser322 within the extracellular domain. L-selectin is also down-modulated from the surface in response to cross-linking, possibly through a similar mechanism. To further characterize the mechanism of down-modulation, several L-selectin mutants were generated and transfected into COS cells. Wild-type L-selectin as well as mutants with one or two amino acid substitutions at the cleavage site were nearly quantitatively shed into the culture supernatant. However, mutants in which a nine-amino acid stretch that included the protease-sensitive site was either deleted or replaced with a polyglycine spacer or a comparable region of E-selectin were retained on the cell surface and not detected in the supernatant. These results are consistent with other reports describing protease resistant L-selectin mutants. We also demonstrate that when expressed in L1-2 pre-B cells, the L-selectin nine-amino acid deletion mutant (321del.9), but not wild-type L-selectin, is resistant to down-regulation induced by PMA. However, both wild-type and mutant 321del.9 are completely lost from the cell surface in response to cross-linking with an L-selectin Ab. PMA-induced- but not L-selectin cross-linking-induced down-modulation was inhibited by staurosporine. These data are consistent with the idea that the L-selectin protease(s) can tolerate minor structural alterations at the cleavage site, and that L-selectin down-modulation can be induced by more than one mechanism, at least one of which (cross-linking) is protein kinase C independent.
authors.affiliation: Division of Hematology-Oncology and Tupper Research Institute, Department of Medicine, New England Medical Center, Tufts University School of Medicine, Boston, MA 02111, USA.
authors.forename: J H
authors.initials: JH
authors.lastname: Stoddart
authors.forename: R R
authors.initials: RR
authors.lastname: Jasuja
authors.forename: M A
authors.initials: MA
authors.lastname: Sikorski
authors.forename: U H
authors.initials: UH
authors.lastname: von Andrian
authors.forename: J W
authors.initials: JW
authors.lastname: Mier
mesh_terms: D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents; D015536:Down-Regulation; D010450:Endopeptidases / Q000378:metabolism; D006801:Humans; D007158:Immunologic Techniques; D019041:L-Selectin / Q000235:genetics / Q000378:metabolism*; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D003432:Cross-Linking Reagents; D019041:L-Selectin; D010450:Endopeptidases; D013755:Tetradecanoylphorbol Acetate