Differential expression and ligand binding properties of tumor necrosis factor receptor chimeric mutants.
The receptors for tumor necrosis factor (TNF) are represented by two transmembrane proteins, p55TNFR and p75TNFR, which are members of a family of cell surface molecules, including the Fas antigen, CD30, CD40, OX40, a Shope fibroma virus protein, and the low affinity p75 nerve growth factor receptor. A common structural feature is a sequence of 40 amino acids that is found in adjacent repeated domains, with 6 cysteine residues in a conserved register. To assess the functional significance of this cysteine-rich domain (CRD), we have constructed chimeric receptors between each TNF receptor and the low affinity nerve growth factor receptor. The chimeric receptor cDNAs were expressed efficiently in COS-1 and 3T3 fibroblasts, as assessed by affinity cross-linking, cell surface biotinylation and immunoprecipitation, and equilibrium binding. Receptors with two CRD of either TNF receptor were incapable of binding TNF, whereas receptors with all four CRD retained the ability to bind TNF with wild type affinity. These results, in conjunction with previous deletion mutation studies, suggest that TNF binding to each receptor requires all four cysteine-rich repeats. Furthermore, analysis of chimeric receptors containing domains of p55TNFR suggests that cytoplasmic sequences directly influence the levels of receptor expression.
Hsu
K C
KC
Department of Cell Biology and Anatomy, Cornell University Medical College, New York, New York 10021.
Chao
M V
MV
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003545:Cysteine / Q000378:metabolism; D004247:DNA; D000882:Haplorhini; D006801:Humans; D015151:Immunoblotting; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D008024:Ligands; D011956:Receptors, Cell Surface; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D004247:DNA; D003545:Cysteine