TNF Recruits TRADD to the Plasma Membrane But Not the trans -Golgi Network, the Principal Subcellular Location of TNF-R1

Abstract

The subcellular localization of TNF-R1 to the Golgi apparatus, initially observed in endothelial cells, has been confirmed using transfection of bovine aortic endothelial cells with a human TNF-R1 expression plasmid. The subcellular interactions of TNF-R1 and the TRADD (TNFR-associated death domain protein) adaptor protein have been analyzed in the human monocyte cell line U937 and the human endothelial cell line ECV304 by confocal immunofluorescence microscopy and by Western blot analysis of fractionated cell extracts. In untreated cells, in which TNF-R1 is found on the cell surface but principally localizes to the trans-Golgi network, TRADD is concentrated in the cis- or medial-Golgi region, but separates from the Golgi during cell fractionation. Coimmunoprecipitation studies have shown that TRADD binds to TNF-R1 within 1 min of TNF treatment in a cell fraction-containing plasma membrane. This association is followed by a gradual dissociation, which is prevented if receptor-mediated endocytosis is inhibited by hypertonic medium. In contrast, no association is detected between TRADD and TNF-R1 in the Golgi in response to exogenous TNF at any time examined. These results suggest that although TNF-R1 is predominantly a Golgi-associated protein and TRADD also localizes to the Golgi region, exogenous TNF causes TRADD to bind to TNF-R1 only at the plasma membrane.