CHARACTERIZATION OF A RECOMBINANT EXTRACELLULAR DOMAIN OF THE TYPE-1 TUMOR-NECROSIS-FACTOR RECEPTOR - EVIDENCE FOR TUMOR-NECROSIS-FACTOR-ALPHA INDUCED RECEPTOR AGGREGATION

An expression plasmid encoding the extracellular portion of the human tumor necrosis factor (TNF) type 1 receptor (TNF-R1) was constructed and used to generate a stable cell line secreting soluble TNF-R1 (sTNF-R1). The sTNF-R1 was purified, and its biochemical properties and its interactions with human TNF-alpha were examined. SDS-PAGE resolved the purified sTNF-R1 into three bands of approximate M(r) 24 200, 28 200, and 32 800. Sedimentation equilibrium analysis gave a molecular weight of 25 000 for sTNF-R1 whereas the molecular weight obtained by gel filtration chromatography was approximately 55 000-60 000. Scatchard analysis of [I-125]TNF-alpha binding to sTNF-R1 revealed high-affinity binding (K(d) = 93 pM), comparable to that observed for the intact receptor on whole cells. Competitive binding experiments showed that sTNF-R1 has a 50-60-fold higher affinity for TNF-alpha than for TNF-beta, in contrast to the equal affinities of TNF-alpha and TNF-beta for the full-length TNF-R1 transiently expressed in mammalian cells. The sTNF-R1 was found to block the cytotoxicity of TNF-alpha and TNF-beta on a murine L-M cell assay. The sizes of the sTNF-R1.TNF-alpha complex determined by gel filtration chromatography and sedimentation equilibrium were approximately 141 and 115 kDa, respectively. The stoichiometry of the complex was examined by Scatchard analysis, size-exclusion chromatography, HPLC separation, amino acid composition, sequence analysis, and sedimentation equilibrium. The data from these studies suggest that at least two molecules of sTNF-R1 can bind to a single TNF-alpha trimer. We propose that the initiation of signaling by TNF-R1 involves TNF-alpha-induced receptor oligomerization.