CHARACTERIZATION OF LIGAND-BINDING BY THE HUMAN P55 TUMOR-NECROSIS-FACTOR RECEPTOR - INVOLVEMENT OF INDIVIDUAL CYSTEINE-RICH REPEATS

Two soluble tumour-necrosis-factor-alpha(TNF)-binding proteins are der ived from the extracellular domains of the p55 and p75 TNF receptors. They are considered to play a pivotal regulatory role in TNF-mediated inflammatory processes, including diseases such as rheumatoid arthriti s, by competing with the cell surface receptors for TNF and lymphotoxi n (LT, tumour-necrosis factor beta). The extracellular domains of the two receptors each contain four similar cysteine-rich repeats of about 40 amino acids, in common with several other cell surface proteins in cluding the p75 nerve-growth-factor receptor and the CD40 and Fas anti gens. The aim of this study was to characterize the involvement of the four cysteine-rich repeats of the human p55 TNF receptor in TNF and L T binding by both membrane-bound and soluble forms of the receptor. In dividual repeats were system atically deleted by PCR mutagenesis and t he variants transiently expressed in COS cells. Immuno-precipitated re ceptor variants exhibited the expected sizes on SDS/PAGE gels, and bou nd a panel of conformation-dependent anti-(TNF receptor) antibodies. B inding of TNF by the four soluble derivatives was compared with bindin g by the wild-type soluble receptor using a TNF-afFinity column and a BIAcore(TM) Biosensor, by measurement of their ability to inhibit TNF cytotoxicity on WEHI cells, and I-125-TNF binding to U937 cells. Delta 4, which lacks the fourth cysteine-rich repeat, bound TNF comparably with the full-length soluble receptor. TNF-binding affinity was unalte red by deletion of the fourth membrane-proximal cysteine-rich repeat, as determined by Scatchard analysis of the transmembrane derivatives. We conclude that the fourth cysteine-rich repeat is not required for T NF binding. In contrast, both the soluble and the transmembrane deriva tives lacking any one of the first, second or third repeats failed to bind TNF. Although we cannot entirely exclude the possibility that thi s may be due to indirect conformational change, rather than the remova l of essential epitopes, our results suggest that the first three repe ats are each required for TNF binding by both the soluble and the cell -surface receptor.