TNF recruits TRADD to the plasma membrane but not the trans-Golgi network,the principal subcellular location of TNF-R1

The subcellular localization of TNF-R1 to the Golgi apparatus, initially ob served in endothelial cells, has been confirmed using transfection of bovin e aortic endothelial cells with a human TNF-R1 expression plasmid, The subc ellular interactions of TNF-RI 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 immunofluoresc ence microscopy and by Western blot analysis of fractionated cell extracts. In untreated cells, in which TNF-R1 is found on the cell surface but princ ipally localizes to the trans-Golgi network, TRADD is concentrated in the c is- or medial-Golgi region, but separates from the Golgi during cell fracti onation, Coimmunoprecipitation studies have shown that TRADD binds to TNF-R 1 within 1 min of TNF treatment in a cell fraction-containing plasma membra ne. This association is followed by a gradual dissociation, which is preven ted 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 th at although TNF-R1 is predominantly a Golgi-associated protein and TRADD al so localizes to the Golgi region, exogenous TNF causes TRADD to bind to TNF -R1 only at the plasma membrane.