DIMERIZATION OF CHIMERIC ERYTHROPOIETIN 75 KDA TUMOR-NECROSIS-FACTOR (TNF) RECEPTORS TRANSDUCES TNF SIGNALS - NECESSITY FOR THE 75 KDA TNF-RECEPTOR TRANSMEMBRANE DOMAIN

We developed a transfection-based assay for evaluating human (h) Tumour Necrosis Factor receptor (TNF-R) activities in a rat/mouse T-cell hybridoma, viz, PC60, Here we report on the role of TNF-R75 cross-linking in induction of GM-CSF secretion and apoptosis. The effect of TNF-R75 dimerization, in contrast to trimerization, was analysed by replacing the extracellular domain of this receptor with the equivalent domain of the murine erythropoietin receptor (EPO-R), which dimerizes upon ligand interaction. To determine the role of the transmembrane region in signal transduction, chimeric EPO-R/TNF-R75 were constructed in which the respective transmembrane domains were interchanged. The hybrid receptors were introduced into PC60hTNFR55 cells, which already expressed functional, transfected hTNF-R55. By this approach we demonstrated that dimerized chimeric EPO-R/TNF-R75 receptors act synergistically with hTNF-R55-induced cytokine production and apoptosis as does trimerized wild-type hTNF-R75, Dimeric triggering of these hybrid receptors with EPO alone was less efficient than trimerization of hTNF-R75, Furthermore, EPO-R/TNFR75 only responded to EPO when the matching transmembrane region of TNF-R75 was present. Our results also prove that the hTNF-R75 extracellular part per se is not required for signalling. Finally, our data indicate that the expression of chimeric EPO-R/TNF-R75 in PC60hTNF-R55 cells, regardless of the presence of the TNF-R75 transmembrane region, facilitates TNF-R55-dependent signal transduction leading to apoptosis. This means that introduction of the intracellular domain of hTNF-R75, even without triggering, is sufficient to promote hTNF-R55-dependent activities in PC60 cells. (C) 1995 Academic Press Limited.