The Brefeldin A-inhibited Guanine Nucleotide-exchange Protein, BIG2, Regulates the Constitutive Release of TNFR1 Exosome-like Vesicles

Aminul Islam,Xiaoyan Shen,Toyoko Hiroi,Joel Moss,Martha Vaughan,Stewart J Levine

doi:10.1074/jbc.m607122200

Abstract

The type I, 55-kDa tumor necrosis factor receptor (TNFR1) is released from cells to the extracellular space where it can bind and modulate TNF bioactivity. Extracellular TNFR1 release occurs by two distinct pathways: the inducible proteolytic cleavage of TNFR1 ectodomains and the constitutive release of full-length TNFR1 in exosome-like vesicles. Regulation of both TNFR1 release pathways appears to involve the trafficking of cytoplasmic TNFR1 vesicles. Vesicular trafficking is controlled by ADP-ribosylation factors (ARFs), which are active in the GTP-bound state and inactive when bound to GDP. ARF activation is enhanced by guanine nucleotide-exchange factors that catalyze replacement of GDP by GTP. We investigated whether the brefeldin A (BFA)-inhibited guanine nucleotide-exchange proteins, BIG1 and/or BIG2, are required for TNFR1 release from human umbilical vein endothelial cells. Effects of specific RNA interference (RNAi) showed that BIG2, but not BIG1, regulated the release of TNFR1 exosome-like vesicles, whereas neither BIG2 nor BIG1 was required for the IL-1beta-induced proteolytic cleavage of TNFR1 ectodomains. BIG2 co-localized with TNFR1 in diffusely distributed cytoplasmic vesicles, and the association between BIG2 and TNFR1 was disrupted by BFA. Consistent with the preferential activation of class I ARFs by BIG2, ARF1 and ARF3 participated in the extracellular release of TNFR1 exosome-like vesicles in a nonredundant and additive fashion. We conclude that the association between BIG2 and TNFR1 selectively regulates the extracellular release of TNFR1 exosome-like vesicles from human vascular endothelial cells via an ARF1- and ARF3-dependent mechanism.

Highlights

Tumor necrosis factor (TNF)2 is an important regulator of inflammation, apoptosis, and innate immune responses
TNFR1 exosome-like vesicles were initially identified in conditioned medium from human umbilical vein endothelial cells (HUVEC), which contained 20 –50-nm exosome-like vesicles that were pelleted by high speed centrifugation, sedimented to a density of 1.1 g/ml, and were capable of binding TNF [16]
We showed that calcium-dependent formation of a complex comprising ARTS-1, a type II integral membrane aminopeptidase, and NUCB2, a putative DNA- and calcium-binding protein, associates with cytoplasmic TNFR1 prior to the commitment of TNFR1 to Small interfering RNA duplexes (siRNA), small interfering RNA duplexes; TGN, trans-Golgi network; TNFR1, Type I 55-kDa TNF receptor (TNFRSF1A); PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; WT, wild type

Summary

Introduction

Tumor necrosis factor (TNF) is an important regulator of inflammation, apoptosis, and innate immune responses. The first involves the proteolytic cleavage of TNFR1 ectodomains by a receptor sheddase that results in the shedding of soluble TNFR1 ectodomains [5,6,7,8,9]. BIG2 Regulates TNFR1 Exosome-like Vesicle Release pathways that result in either the constitutive release of TNFR1 exosome-like vesicles or the inducible proteolytic cleavage of TNFR1 ectodomains [22, 23]. Taken together, these findings suggested the probable involvement of intracytoplasmic vesicular trafficking between ERGolgi and cell surface plasma membranes in these processes. The experiments reported here demonstrate that BIG2, but not BIG1, was required for the constitutive release of full-length TNFR1 in exosome-like vesicles from human vascular endothelial cells

Methods

Results

Conclusion