Characterization of the receptor for lymphotoxin; a spontaneous internalization without recycling and ligand-induced downregulation in HL-60 cells.
The receptor system for lymphotoxin (LT) was investigated by use of the human promyelocytic HL-60 cell line. Utilizing subcellular fractionation, internalization of cell surface bound recombinant LT (rLT) and transfer to lysosomes followed by degradation was demonstrated. Binding of rLT to its cell surface receptor induced a downregulation of the receptor which was persistent for at least 6 h. Downregulation of the receptors from the cell surface by activation of protein kinase C with the diacylglycerol OAG was completely reversible but the recovery of the binding capacity was dependent on protein synthesis as it was inhibited by cycloheximide. Treatment with cycloheximide alone resulted in a loss of binding capacity with a half life of approximately 2 h, suggesting a spontaneous consumption of receptors. Affinity cross-linking revealed three ligand-receptor complexes with Mr of 85, 105 and 125 kDa. Because of a strong tendency for ligand polymerization these results suggest oligomer binding of the ligand to a single receptor molecule with an apparent Mr of 70 kDa. N-linked glycosylation constituted 4 to 5 kDa of the total molecular weight. In conclusion, we demonstrated a spontaneous internalization of the receptor for LT without recycling, and that ligand binding resulted in an irreversible downregulation of the receptor.
Gullberg
U
U
Department of Medicine, University of Lund/Sweden.
Lantz
M
M
Nilsson
E
E
Olsson
I
I
D001216:Asparagine / Q000032:analysis; D002458:Cell Fractionation; D002460:Cell Line; D003432:Cross-Linking Reagents; D004075:Diglycerides / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006031:Glycosylation; D006801:Humans; D008024:Ligands / Q000378:metabolism; D008233:Lymphotoxin-alpha / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D011971:Receptors, Immunologic / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D003432:Cross-Linking Reagents; D004075:Diglycerides; D008024:Ligands; D008233:Lymphotoxin-alpha; D008565:Membrane Proteins; D011971:Receptors, Immunologic; D001216:Asparagine; C038237:1-oleoyl-2-acetylglycerol; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate