Receptor-mediated Endocytosis Is Not Required for Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-induced Apoptosis

Susan L Kohlhaas,Andrew Craxton,Xiao-Ming Sun,Michael J Pinkoski,Gerald M Cohen

doi:10.1074/jbc.m700438200

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.

Highlights

Since the recognition of the death-inducing signaling complex (DISC) it has generally been considered that activation of the extrinsic pathway occurred almost exclusively at the plasma membrane
Induction of apoptosis by CD95 seems to occur by two main routes, 1) in type I cells where it involves formation of large amounts of DISC accompanied by rapid receptor internalization and does not require a mitochondrial amplification loop or pathway and 2) in type II cells, which form a DISC more slowly and less extensively and require amplification through crosstalk with mitochondria [15, 16]
Because TNF-related apoptosis-inducing ligand (TRAIL) internalization was partially blocked in HeLa cells expressing the K44A dynamin mutant, we investigated the role of clathrin-dependent endocytosis in TRAIL-induced apoptosis

Summary

Introduction

Since the recognition of the DISC it has generally been considered that activation of the extrinsic pathway occurred almost exclusively at the plasma membrane. Ligand Internalization Is Not Required for TRAIL-mediated tions of z-VAD-fmk (10 ␮M), a broad spectrum caspase inhibitor, Cell Death—To address the possible role of TRAIL internaliza- completely inhibited the induction of cell death assessed by these tion in the induction of apoptosis, soluble TRAIL was adhered three different criteria (Table 1).

Results

Conclusion