Coexpression of the human TNF receptors TR60 and TR80 in insect cells: analysis of receptor complex formation.

For investigation of a possible physical interaction between the two human tumor necrosis factor receptors, TR60 (type I) and TR80 (type II), the baculovirus expression system was used. Each of the receptors was expressed as a membrane-integrated protein in insect cells, able to specifically bind the two ligands, tumor necrosis factor (TNF) and lymphotoxin (LT alpha). Typically, about 150,000 membrane receptors per cell could be detected 40 h after infection, exerting high affinity ligand binding capacity with Kd values virtually identical to that of human cell lines. The baculovirus system allowed coexpression of both TNF membrane receptors at very high and about equal numbers to investigate the existence of heteromultimeric receptor complexes, either formed spontaneously or ligand induced. Neither saturation binding studies nor immunoprecipitation experiments gave an indication for the existence of TNF receptor heteromers. These data are in accordance with the current view of TNF signaling, in which homonultimerization, rather than heteromer formation of TNF receptors is the initial activating event.