Structural and functional analysis of the two human tumor necrosis factor receptors

Paul Chih-Hsueh Chen, Thomas Jefferson University

Abstract

Pleiotropic TNF activities are mediated through the two cell surface receptors (TNFRs). Both are transmembrane proteins sharing 30% protein sequence homology in their extracellular domains, but little homology in intracellular domains. It suggests that the two receptors interact with separate signal molecules which mediate distinct biological activities. The extracellular domain of TNFRs are characterized by 4 conserved cysteine-rich motif repeat domains. In order to identify the ligand-binding domain(s) of the two TNFRs, recombinant domain-deletion mutants of TNFRs were expressed and their binding capacities assessed by ligand blot analysis and Scatchard analysis. Our results are consistent with those from x-ray cinematography, that domain 3 of both TNFRs is critical for TNF-$\alpha$ binding. But the domain 4 of TNFR-2, but not that of TNFR-1, is also important for binding. Whether the domain 4 of TNFR-2 is involved directly in binding TNF, or indirectly in the correct folding of other domains of the receptor, is still unknown. To investigate the functional species specificity of TNFR-1, we transfected an inducible hTNFR-1 expression construct into murine L929 cells. These established L929 cells, are about as sensitive to hTNF-$\alpha$ as parental cells, in the non-induced state. However, they become 25-100 fold more sensitive to hTNF-$\alpha$ after hTNFR-1 expression is induced. $\sp{125}$I-TNF-$\alpha$ saturation binding assay and Scatchard analysis showed that the transfected hTNFR-1, interacts with hTNF-$\alpha$ much more strongly than mTNFR-1 does. Taken together, these results demonstrate homologous species preference of TNFR-1. The advantage of inducible hTNFR-1 expression and the significance of these cell lines on clinical research for sensitive and reproducible hTNF bioassay are discussed. The functional roles of the two TNFRs in mediating NF-$\kappa$B activation, and inducing IL-2R$\alpha$ chain (CD25) and ICAM-1 (CD54) expression, were examined using receptor-specific antisera and TNF mutants, Northern blot analysis, gel shift assay and flow cytometry on human NK-like YT cells. The results showed that antisera against the two hTNFRs and human TNF mutants binding exclusively or preferentially to one of the two TNFRs are all capable of triggering NF-$\kappa$B activation and CD25/CD54 nduction. In combination, the two receptor antisera produce synergistic responses. Northern blot analyses indicated that the mRNAs of p50 and p52, but not that of p65 RelA, are induced by TNF or either of the two TNFR antisera. Supershift assay identified that the nuclear NF-$\kappa$B complexes activated by TNF are P50/p50 and p50/p65 dimers. Finally, a TNF triggered homoaggregation on YT cells was observed. We showed that TNFR-2 mediates this phenomenon much stronger than TNFR-1 does. Taken together, these results indicated that NF-$\kappa$8 activation, homoaggregation, and the induction of CD25 and CD54 on YT cells could be mediated independently through individual TNFRs via separate signaling pathways. Finally, by successful identification of TNF-induced genes, including MCP-1 (monocyte chemoattractant protein-1), in human monocyrtic leukemia cells ML-1a using a modified differential display technique, we showed that this new technique is a powerful tool in identifying TNF-regulated genes involved in different biological activities.

Subject Area

Molecular biology

Recommended Citation

Chen, Paul Chih-Hsueh, "Structural and functional analysis of the two human tumor necrosis factor receptors" (1997). ETD Collection for Thomas Jefferson University. AAI9703675.
https://jdc.jefferson.edu/dissertations/AAI9703675

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