Mechanisms of TNF receptor action: Studies using chimeric receptor mutants

The pleiotropic effects of tumor necrosis factor (TNF) appear to be mediated by two distinct receptor subtypes, p55 and p75. Both receptors have extracellular domains containing four 40-amino acid repeats with six cysteine residues at highly conserved positions. The cysteine-rich motif has defined a growing family of transmembrane molecules, which includes both TNF receptors, the p75 low-affinity NGF receptor, the Fas antigen, CD40, CD27, OX4O, and CD30. We have constructed chimeric molecules between each TNF receptor and the p75 low-affinity NGF receptor to study the role of the cysteine-rich domain (CRD) in ligand binding. Based on affinity crosslinking and Scatchard analysis, it was determined that all four CRDs are required for ligand binding, implying multiple contacts between receptor and ligand.

Provision of specific anti-p55 and anti-p75 antisera made it possible to demonstrate association of the two receptor subtypes by affinity crosslinking and coimmunoprecipitation. Use of the chimeric receptors allowed us to determine that the TNF binding domain is critical to this association, but that the transmembrane and cytoplasmic regions are not. Furthermore, cell-surface biotinylation and immunoprecipitation experiments indicated that receptor aggregation occurs only in the presence of ligand.

Finally, the function of the p75 TNF receptor was assessed by examining the effect of receptor expression on TNF responses such as cell death and NF-$\kappa$B activation. While p75 does not seem to play a role in signaling TNF-mediated cytotoxicity, it is capable of activating the transcription factor NF-$\kappa$B in a ligand-independent and p55-independent fashion. Expression of various p75 chimeric receptor mutants indicated that the presence of an intact p75 cytoplasmic domain is sufficient for NF-$\kappa$B activation. This work clearly establishes a role for p75 in activating a signal transduction pathway central to TNF stimulation of gene expression.