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 Mr 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 [125I]TNF-alpha binding to sTNF-R1 revealed high-affinity binding (Kd = 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Pennica
D
D
Department of Molecular Biology, Genentech, Inc., South San Francisco, California 94080.
Kohr
W J
WJ
Fendly
B M
BM
Shire
S J
SJ
Raab
H E
HE
Borchardt
P E
PE
Lewis
M
M
Goeddel
D V
DV
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002850:Chromatography, Gel; D003601:Cytotoxicity Tests, Immunologic; D006801:Humans; D051379:Mice; D008970:Molecular Weight; D011940:Receptor Aggregation; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification; D012995:Solubility; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D014461:Ultracentrifugation
D011956:Receptors, Cell Surface; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha