CHIMERIC TUMOR-NECROSIS-FACTOR RECEPTORS WITH CONSTITUTIVE SIGNALING ACTIVITY

Many hormone and cytokine receptors are crosslinked by their specific ligands, and multimerization is an essential step leading to the gener ation of a signal, In the case of the tumor necrosis factor (TNF) rece ptors (TNF-Rs), antibody-induced crosslinking is sufficient to trigger a cytolytic effect, However, the quaternary structural requirements f or signaling-i.e., the formation of dimers, trimers, or higher-order m ultimers-hale remained obscure, Moreover, it has not been clear whethe r the 55-kDa or 75-kDa TNF-R is responsible for initiation of cytolysi s, We reasoned that an obligate receptor dimer, targeted to the plasma membrane, might continuously signal the presence of TNF despite the a ctual absence of the ligand, Such a molecule, inserted into an appropr iate vector, could be used to project receptor-specific ''TNF-like'' a ctivity to specific cells and tissues in vivo, Accordingly, we constru cted sequences encoding chimeric receptors in which the extracellular domain of the mouse erythro-poietin receptor (Epo-R) was fused to the ''stem,'' transmembrane domain, and cytoplasmic domain of the two mous e TNF-Rs, Thus, the Epo-R group was used to drive dimerization of the TNF-R cytoplasmic domain, These chimeric proteins were well expressed in a variety of cell lines and bound erythropoietin at the cell surfac e, Both the 55-kDa and the 75-kDa Epo/TNF-R chimeras exerted a constit utive cytotoxic effect detected by cotransfection or clonogenic assay, Thus, despite the lack of structural homology between the cytoplasmic domains of the two TNF-Rs, a similar signaling endpoint was observed. Moreover, dimerization (rather than trimerization or higher-order mul timerization) was sufficient for elicitation of a biological response.