Dimerization of chimeric erythropoietin/75 kDa tumour 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.
Declercq
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Laboratory of Molecular Biology, Gent University, Belgium.
Vandenabeele
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Fiers
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D000818:Animals; D015703:Antigens, CD / Q000737:chemistry*; D017209:Apoptosis / Q000502:physiology; D001483:Base Sequence; D001704:Biopolymers; D004921:Erythropoietin / Q000502:physiology; D005069:Evaluation Studies as Topic; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D006825:Hybridomas; D051379:Mice; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D051381:Rats; D017467:Receptors, Erythropoietin / Q000737:chemistry*; D018124:Receptors, Tumor Necrosis Factor / Q000737:chemistry*; D047889:Receptors, Tumor Necrosis Factor, Type II; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection / Q000502:physiology
D015703:Antigens, CD; D001704:Biopolymers; D017467:Receptors, Erythropoietin; D018124:Receptors, Tumor Necrosis Factor; D047889:Receptors, Tumor Necrosis Factor, Type II; D011993:Recombinant Fusion Proteins; D004921:Erythropoietin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor