L-selectin shedding is independent of its subsurface structures and topographic distribution.
L-selectin (CD62L), a lectin-like adhesion molecule, mediates lymphocyte homing and leukocyte accumulation at sites of inflammation. Its transmembrane (TM) and intracellular (IC) domains confer clustering of L-selectin on microvilli of resting leukocytes, which is important for L-selectin function. Following activation of protein kinase C (PKC) or calmodulin inhibition, the wild-type (WT) protein is rapidly cleaved in its membrane-proximal ectodomain. To examine whether L-selectin topography or TM/IC domains are involved in this shedding process, we used stable transfectants expressing WT L-selectin (on microvilli) or chimeric molecules consisting of the L-selectin ectodomain linked to the TM/IC domains of CD44 (excluded from microvilli) or CD31 (randomly distributed). PKC activation by PMA altered the cells' surface morphology, but did not induce a redistribution of L-selectin ectodomains. All cell lines shed ectodomains upon PMA activation in a dose-dependent fashion and with similar kinetics. Calmodulin inhibition by trifluoperazine induced shedding in both WT and chimera transfectants. At high trifluoperazine concentrations, shedding of WT L-selectin was significantly more pronounced than that of chimeric molecules. Regardless of the activating stimulus, shedding was blocked by a hydroxamate-based metalloprotease inhibitor, suggesting that ectodomain down-regulation occurred through proteolytic cleavage by identical protease(s). These results show that the recognition site(s) for PKC-induced L-selectin shedding is exclusively contained within the ectodomain; the nature of subsurface structures and surface topography are irrelevant. Shedding induced by calmodulin inhibition has two components: one requires the L-selectin TM/IC domain, and the other is independent of it.
Fors
B P
BP
Center for Blood Research and Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA.
Goodarzi
K
K
von Andrian
U H
UH
D000818:Animals; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002460:Cell Line; D004151:Dipeptides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006877:Hydroxamic Acids / Q000494:pharmacology; D007700:Kinetics; D019041:L-Selectin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007962:Leukocytes / Q000276:immunology; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors; D051379:Mice; D008871:Microvilli / Q000648:ultrastructure; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014268:Trifluoperazine / Q000494:pharmacology
D002147:Calmodulin; D004151:Dipeptides; D004791:Enzyme Inhibitors; D006877:Hydroxamic Acids; C099147:KD-IX 73-4; D011993:Recombinant Fusion Proteins; D019041:L-Selectin; D014268:Trifluoperazine; D008666:Metalloendopeptidases; D013755:Tetradecanoylphorbol Acetate