Abstract
To investigate the signaling mechanism of the 55-kDa tumor necrosis factor (TNF) receptor a functional transfection based assay was developed. The human 55-kDa TNF receptor, stably expressed in mouse L929 cells, was demonstrated to be activated specifically by agonist antibodies and to initiate a signal for cellular cytotoxicity. A deletion mutant of the human TNF receptor lacking most of the cytoplasmic domain was found to be completely defective in generating the signal for cytotoxicity. Additionally, expression of the truncated receptor substantially suppressed signaling by endogenous mouse TNF receptors in response to TNF, but not in response to specific anti-murine TNF receptor antibodies. These results suggest that aggregation of 55-kDa TNF receptor intracellular domains, which are not associated in the absence of ligand, is an important component of the signal for cellular toxicity. This work also provides an example of a dominant negative mutation in a transmembrane receptor that lacks a tyrosine kinase domain, and suggests a more general utility of dominant negative mutations in the investigation of cytokine receptor function.
Highlights
To investigate the signaling mechanism of the 66kDa tumor necrosis factor (TNF) receptor a functional transfection based assay was developed
The murine TNF-R1 was shown to be responsible for signalingcytotoxicity and theinduction of several genes, while the murine TNF-R2 was shown to be capable of signaling proliferation of primary thymocytes and a cytotoxic T cell line [24]
The signaling mechanism of neither TNF receptor is understood, several studies have suggested that cross-linking of receptor extracellular domains by either TNF or agonist antibodies is an important feature of the activation process of TNF-R1 [15, 25, 26]
Summary
A DOMINANT NEGATIVE MUTATION SUPPRESSES THE ACTIVATION OF THE 55-KDA TUMOR NECROSIS FACTOR RECEPTOR*. To investigate the signaling mechanism of the 66kDa tumor necrosis factor (TNF) receptor a functional transfection based assay was developed. The human 66-kDa TNF receptor, stably expressed in mouse L929 cells, was demonstrated to be activated by agonist antibodies and to initiate a signal for cellular cytotoxicity. A number of reports have described studies investigating the individual roles of the two TNF receptors Both polyclonal and monoclonal antibodies directed against human TNF-R1 have been shown to behave as receptor agonists and elicit several TNF activities such as cytotoxicity, fibroblast proliferation, resistance to chlamydiae, and synthesis of prostaglandin E2 [15, 22, 23]. In an attempt tocircumvent this problem, we have chosen to express the human 55-kDa TNF receptor in the highly TNF-sensitive murine cell line L929, and activate the transfectedhuman TNF receptor with species-specificagonist antibodies.
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