Mapping the domain(s) critical for the binding of human tumor necrosis factor-alpha to its two receptors.
The extracellular domains of the two human tumor necrosis factor (TNF) receptors critical for binding TNF-alpha were examined by deletion mapping. The ligand binding capability of full-length and truncated recombinant soluble TNF receptors (TNFRs) was assessed by ligand blot analysis and their binding affinity determined by Scatchard analysis. The results showed that deletion of the fourth cysteine-rich domain of the p55 receptor (TNFR-1) did not alter ligand binding affinity significantly. Deletion of domains 3 and 4 of TNFR-1 resulted in no ligand binding, suggesting that domain 3, but not 4, of TNFR-1 binds directly to ligand. Deletion of domain 4 of TNFR-2 resulted in drastically reduced protein yield and 3-fold reduction in ligand binding affinity, while deletion of both domains 4 and 3 yielded no protein. Thus, the domain 4 of TNFR-2, but not that of TNFR-1, appears to be involved directly in binding TNF, although it is also possible that the domain 4 of TNFR-2 is involved in the correct folding of other domains. These results suggest that the modes of interaction between TNF-alpha and its dual receptors are different, providing opportunity to modulate each receptor specifically for research and therapeutic purposes.
Chen
P C
PC
Department of Microbiology, Thomas Jefferson University, Jefferson Medical College, Philadelphia, Pennsylvania 19107.
DuBois
G C
GC
Chen
M J
MJ
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000235:genetics; D010449:Peptide Mapping; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism*; D017384:Sequence Deletion; D018411:Spodoptera; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*
D004274:DNA, Recombinant; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha