Abstract 222 Cytokines: Intracellular Signaling Platform, Tuesday, 5/4

Background: Vascular endothelial cells (ECs), a major target of TNF action, express both the 55kD (TNFR1) and 75kD (TNFR2) TNF receptors. Most of the effects of TNF, including gene activation and cell death, are mediated by TNFR1. A region of the intracellular portion of TNFR1 called the "death domain" (DD) recruits the adaptor protein TRADD to initiate TNF signal transduction. Although these interactions are thought to occur at the plasma membrane, TNFR1 in ECs is predominantly expressed in the Golgi apparatus. TNFR2 does not contain a DD and is more highly expressed on the cell surface. The relationship between receptor localization, structure, and function is not clear. We examined whether the DD, required for TNF signaling through TNFR1, is necessary for receptor localization.

Methods: Human umbilical vein endothelial cells (HUVEC) were transiently transfected with various expression constructs encoding TNFR1 or TNFR2, including full-length TNFR1, TNFR1 minus the complete intracellular domain (TNFR1-IC), TNFR1 minus the death domain (TNFR1-DD), full-length TNFR2, and a chimera consisting of full-length TNFR2 plus the DD from TNFR1 (TNFR2+DD). These recombinant receptors were epitope-tagged with FLAG or linked to green fluorescent protein (GFP). Transfected cells were examined by immunofluorescence and confocal microscopy, as well as by FACS analysis.

Results: TNFR1 transfected into HUVEC was expressed in the perinuclear region and co-localized with anti-mannosidase to the Golgi, consistent with the localization pattern of endogenous TNFR1. This cellular distribution was seen with the FLAG-tagged and GFP-fusion constructs; full-length TNFR1 was not detectable on the cell surface by microscopy or FACS. Removal of the DD in deletion constructs TNFR1-IC and TNFR1-DD allowed expression of the receptor on the cell surface. Transfected TNFR2 was expressed on the cell surface and intracellularly, similar to the distribution of endogenous TNFR2. Inclusion of the DD in the chimera TNFR2+DD did not alter this localization pattern, i.e., did not cause Golgi retention.

Conclusion: The death domain of TNFR1 is necessary for Golgi retention of TNFR1 in endothelial cells; removal of the DD allows translocation of the receptor to the cell surface. The addition of the DD to TNFR2 is not sufficient to cause Golgi retention of TNFR2. These results suggest that the DD must interact, directly or indirectly, with other regions of TNFR1 to cause Golgi retention. The spatial segregation of TNFR1 and TNFR2 may regulate receptor interaction and ligand sensitivity.