title: Receptor-mediated endocytosis is not required for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis.
abstract: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is selectively toxic to tumor compared with normal cells. Other members of the TNF family of death ligands (TNF, CD95L) engage their respective receptors (TNF-R1 and CD95), resulting in internalization of receptor and ligand and recruitment of adaptor proteins to the caspase activation platform known as the death-inducing signaling complex (DISC). Recently, TNF-R1 and CD95 have been shown to induce apoptosis with an absolute requirement for internalization of their corresponding receptors in the formation of a DISC. We show that TRAIL and its receptors are rapidly endocytosed in a time- and concentration-dependent manner. Blockade of receptor internalization with hyperosmotic sucrose did not inhibit TRAIL-induced apoptosis but, rather, amplified the apoptotic signaling of TRAIL. Plate-bound and soluble TRAIL induced similar levels of apoptosis. Together these results suggest that neither ligand nor receptor internalization is required for TRAIL-induced apoptosis. Internalization of TRAIL is mediated primarily by clathrin-dependent endocytosis and also by clathrin-independent pathways. Inhibition of clathrin-dependent internalization by overexpression of dominant negative forms of dynamin or AP180 did not inhibit TRAIL-induced apoptosis. Consistent with the finding that neither internalization of TRAIL nor its receptors is required for transmission of its apoptotic signal, recruitment of FADD (Fas-associated death domain) and procaspase-8 to form the TRAIL-associated DISC occurred at 4 degrees C, independent of endocytosis. Our findings demonstrate that TRAIL and TRAIL receptor 1/2, unlike TNF-TNF-R1 or CD95L-CD95, do not require internalization for formation of the DISC, activation of caspase-8, or transmission of an apoptotic signal in BJAB type I cells.
authors.affiliation: MRC Toxicology Unit, Hodgkin Building, University of Leicester, P. O. Box 138, Lancaster Road, Leicester LE1 9HN, United Kingdom.
authors.forename: Susan L
authors.initials: SL
authors.lastname: Kohlhaas
authors.forename: Andrew
authors.initials: A
authors.lastname: Craxton
authors.forename: Xiao-Ming
authors.initials: XM
authors.lastname: Sun
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Pinkoski
authors.forename: Gerald M
authors.initials: GM
authors.lastname: Cohen
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics / Q000502:physiology*; D053181:Caspase 8 / Q000378:metabolism; D003080:Cold Temperature; D053418:Death Domain Receptor Signaling Adaptor Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D034281:Dynamins / Q000096:biosynthesis / Q000235:genetics; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D053222:Fas Ligand Protein / Q000378:metabolism / Q000494:pharmacology; D053200:Fas-Associated Death Domain Protein / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000494:pharmacology; D019014:fas Receptor / Q000378:metabolism
chemical_list: D053418:Death Domain Receptor Signaling Adaptor Proteins; C505533:FADD protein, human; D053222:Fas Ligand Protein; D053200:Fas-Associated Death Domain Protein; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D053221:TNF-Related Apoptosis-Inducing Ligand; C105190:TNFRSF10A protein, human; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; D019014:fas Receptor; D053181:Caspase 8; D034281:Dynamins