HUMAN TUMOR-NECROSIS-FACTOR-ALPHA (TNF-ALPHA) MUTANTS WITH EXCLUSIVE SPECIFICITY FOR THE 55-KDA OR 75-KDA TNF RECEPTORS

To probe the ligand receptor interface, a number of point mutations we re introduced in selected regions of human tumor necrosis factor (TNF) alpha by site-directed mutagenesis. The mutated proteins were express ed in Escherichia coli and analyzed for selective binding to recombina nt 55- and 75-kDa TNF receptors in competition with radiolabeled wild- type TNFalpha. Generally, mutations in the loop from position 29 to 34 and at positions 86 and 146 preferentially impaired binding to the 75 -kDa TNF receptor, whereas mutations in the region from 143 to 145 mai nly affected binding to the 55-kDa TNF receptor. Mutation of the conse rved Tyr87 resulted in a dramatic loss of binding activity to both rec eptors. The selectivity for one or the other receptor type was found t o be enhanced by combining two or three point mutations, the effects o f the single mutations with respect to receptor selectivity being at l east additive. A combination of the mutations Arg32-->Trp and Ser86--> Thr yielded a double mutant (R32W-S86T) with wild-type binding to the 55 kDa, but no measurable binding to the 75-kDa TNF receptor. In contr ast, combining the Asp143-->Asn and Ala145-->Arg mutations (D143N-A145 R) resulted in a complete loss of binding to the 55-kDa TNF receptor, whereas binding to the 75-kDa TNF receptor was impaired by only 5-10-f old. In functional assays, selective activation of the 55-kDa TNF rece ptor by the R32W-S86T mutant elicited a full cytotoxic response in hum an KYM-1 cells and secretion of interleukin 6 and granulocyte-macropha te colony-stimulating factor in human umbilical vein endothelial cells . In contrast, stimulation of the 75-kDa TNF receptor with the D143N-A 145R mutant as well as with agonistic antibodies failed to induce thes e responses.