ADAM-17-independent shedding of L-selectin.
L-selectin is expressed by leukocytes and facilitates their adhesion under flow along the walls of blood vessels. As do a variety of membrane proteins, L-selectin undergoes ectodomain shedding. Using approaches that monitor full-length L-selectin in short-term assays, it has been determined that L-selectin shedding is defective in tumor necrosis factor alpha-converting enzyme (ADAM-17)-deficient cells. In this study, we examined the steady-state levels of L-selectin on ADAM-17-deficient cells using a monoclonal antibody to the cytoplasmic region of L-selectin, which allows for the detection of total L-selectin (full-length and the membrane-associated cleavage fragment). We demonstrate that ADAM-17-deficient cells generate a 6-kDa transmembrane fragment of L-selectin. Although inducible L-selectin shedding by phorbol 12-myristate 13-acetate stimulation was not observed by these cells in short-term assays, basal turnover did occur, resulting in the production of soluble L-selectin, as determined by enzyme-linked immunosorbent assay. L-selectin turnover was greatly increased upon ADAM-17 reconstitution. Truncating the juxtamembrane region of L-selectin blocked ADAM-17-independent shedding as did a hydroxymate metalloprotease inhibitor. Together, these findings demonstrate that a metalloprotease activity separate from ADAM-17 can use the cleavage domain of L-selectin. We speculate that separate proteolytic mechanisms of L-selectin shedding may regulate distinct antiadhesive mechanisms, such as inducible shedding for the rapid dissociation of cell-cell interactions and constitutive shedding for the homeostatic maintenance of high serum levels of soluble L-selectin, a potential adhesion buffer.
Walcheck
Bruce
B
Department of Veterinary PathoBiology, University of Minnesota Academic Health Center, St. Paul, MN 55108, USA. walh003@umn.edu
Alexander
Shelia R
SR
St  Hill
Catherine A
CA
Matala
Erik
E
D051722:ADAM Proteins; D000072198:ADAM17 Protein; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism*; D006877:Hydroxamic Acids / Q000378:metabolism; D019041:L-Selectin / Q000737:chemistry / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000172:deficiency / Q000378:metabolism*; D051379:Mice; D011233:Precipitin Tests; D011480:Protease Inhibitors / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D000911:Antibodies, Monoclonal; D006877:Hydroxamic Acids; D011480:Protease Inhibitors; D014409:Tumor Necrosis Factor-alpha; D019041:L-Selectin; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000072198:ADAM17 Protein; C000606463:Adam17 protein, mouse; D013755:Tetradecanoylphorbol Acetate