Molecular mechanisms of L-selectin-induced co-localization in rafts and shedding [corrected].
Leukocyte recruitment to lymph nodes or inflammatory sites is regulated by adhesion and activation. L-selectin (CD62L) is expressed on leukocytes and mediates tethering and rolling of leukocytes on endothelial cells. Upon stimulation L-selectin is down-regulated by proteolytic cleavage but the molecular mechanisms regulating this shedding step are poorly defined. To study intracellular mechanisms, we induced shedding of L-selectin by cross-linking with an immobilized L-selectin antibody (Dreg56) in Jurkat cells. The loss of surface expression was quantitated by flow cytometry and the increase of soluble L-selectin was determined by Western blot analysis. We find that Jurkat and p56(lck)-deficient JCaM1.6 cells released L-selectin to similar extent (18+/-4% and 17+/-3%, respectively) and revealed comparable inhibition with the src-tyrosine kinase inhibitor PP2. Glutathione (GSH), an inhibitor of the neutral sphingomyelinase, PD98059, a MAP-kinase (MAP-K) inhibitor and metalloprotease inhibitors (MPI) (TAPI, Ro 31-9790, and BB-3103) reduced significantly L-selectin-induced shedding by 60-80%. In Jurkat cells, L-selectin was present in Triton X-100 insoluble membrane rafts and was constitutively tyr-phosphorylated. Dreg56 cross-linking enhanced phosphorylation and recruitment of L-selectin into rafts which was significantly decreased by pretreatment of cells with PD98059. We conclude, that the metalloproteinase-mediated cleavage of L-selectin from cell surface is triggered by intracellular signaling pathways that are independent of p56(lck) tyrosine kinase activity, but require other tyrosine kinases and the neutral sphingomyelinase. The cleavage of L-selectin might involve membrane rafts as signaling platform.
Phong
Minh-Chau
MC
Department of Pediatrics, Division of Neonatology, University of Heidelberg, Im Neuenheimer Feld 150, 69120 Heidelberg, Germany.
Gutwein
Paul
P
Kadel
Stephanie
S
Hexel
Klaus
K
Altevogt
Peter
P
Linderkamp
Otwin
O
Brenner
Birgit
B
D000906:Antibodies / Q000494:pharmacology; D002460:Cell Line; D002465:Cell Movement; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D019169:Jurkat Cells; D019041:L-Selectin / Q000032:analysis* / Q000276:immunology / Q000378:metabolism*; D061965:Matrix Metalloproteinase Inhibitors; D021962:Membrane Microdomains / Q000737:chemistry*; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D011480:Protease Inhibitors / Q000494:pharmacology; D013108:Sphingomyelin Phosphodiesterase / Q000037:antagonists & inhibitors / Q000502:physiology; D013601:T-Lymphocytes / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D019061:src-Family Kinases / Q000502:physiology
D000906:Antibodies; D004791:Enzyme Inhibitors; D005419:Flavonoids; D061965:Matrix Metalloproteinase Inhibitors; D011480:Protease Inhibitors; D019041:L-Selectin; D019061:src-Family Kinases; D020928:Mitogen-Activated Protein Kinases; D013108:Sphingomyelin Phosphodiesterase; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one