SUMMARYTumour necrosis factor (TNF)-receptor-associated periodic syndrome (TRAPS) is a hereditary autoinflammatory disorder involving autosomal-dominant missense mutations in TNF receptor superfamily 1A (TNFRSF1A) ectodomains. To elucidate the molecular effects of TRAPSrelated mutations, we transfected HEK-293 cells to produce lines stably expressing high levels of either wild-type (WT) or single mutant recombinant forms of TNFRSF1A. Mutants with single amino acid substitutions in the first cysteine-rich domain (CRD1) were produced both as full-length receptor proteins and as truncated forms lacking the cytoplasmic signalling domain (Dsig). High-level expression of either WT or mutant full-length TNFRSF1A spontaneously induced apoptosis and interleukin-8 production, indicating that the mutations in CRD1 did not abrogate signalling. Consistent with this, WT and mutant full-length TNFRSF1A formed cytoplasmic aggregates that co-localized with ubiquitin and chaperones, and with the signal transducer TRADD, but not with the inhibitor, silencer of death domain (SODD). Furthermore, as expected, WT and mutant Dsig forms of TNFRSF1A did not induce apoptosis or interleukin-8 production. However, whereas the WT full-length TNFRSF1A was expressed both in the cytoplasm and on the cell surface, the mutant receptors showed strong cytoplasmic expression but reduced cell-surface expression. The WT and mutant Dsig forms of TNFRSF1A were all expressed at the cell surface, but a proportion of the mutant receptors were also retained in the cytoplasm and co-localized with BiP. Furthermore, the mutant forms of surface-expressed Dsig TNFRSF1A were defective in binding TNF-a. We conclude that TRAPS-related CRD1 mutants of TNFRSF1A possess signalling properties associated with the cytoplasmic death domain, but other behavioural features of the mutant receptors are abnormal, including intracellular trafficking and TNF binding.

Objective. To investigate the effect of mutations in tumor necrosis factor receptor superfamily 1A (TNFRSF1A) on the ability of the receptors to be cleaved from the cell surface upon stimulation. The mutations we studied are associated with clinically distinct forms of TNF receptor-associated periodic syndrome (TRAPS). We also investigated different cell types within the same form of TRAPS.Methods. The shedding of TNFRSF1A in response to stimulation with phorbol myristate acetate was assessed in leukocytes and dermal fibroblasts from patients with C33Y TRAPS, and in HEK 293 cell lines stably transfected with constructs containing wild-type TNFRSF1A and/or TNFRSF1A mutants identified in TRAPS patients.Results. The shedding of TNFRSF1A differed between cell types within the same form of TRAPS. In particular, dermal fibroblasts, but not leukocytes, from C33Y TRAPS patients demonstrated reduced shedding of TNFRSF1A. Shedding of both wild-type and mutant TNFRSF1A from the transfected HEK 293 cells showed minor differences, but was in all cases induced to a substantial extent.Conclusion. Differences in TNFRSF1A shedding are not purely a function of the TNFRSF1A structure, but are also influenced by other features of genetic makeup and/or cellular differentiation. It is unlikely that a defect in TNFRSF1A shedding per se can fully explain the clinical features that are common to TRAPS patients with different TNFRSF1A mutations.