The individual roles of the murine type 1 and type 2 tumor necrosis factor (TNF) receptors (TNF-R1 and TNF-R2) were investigated utilizing (i) the strong species specificity of TNF-R2 for murine TNF compared to human TNF and (it) agonistic rabbit polyclonal antibodies directed against the individual TNF receptors. Proliferation of mouse thymocytes and the murine cytotoxic T-cell line CT-6 is stimulated by murine TNF but not by human TNF. Consistent with this observation, polyclonal antibodies directed against TNF-R2 induced proliferation in both of these cell types, whereas polyclonal antibodies directed against TNF-R1 had no effect. In contrast, cytotoxicity in murine LM cells (which are sensitive to murine and human TNF) was induced by antibodies against TNF-R1 but not by antibodies against TNF-R2. Also, the steady-state level of manganous superoxide dismutase mRNA in the murine NIH 3T3 cell line was induced by murine TNF, human TNF, and anti-TNF-R1 but not by anti-TNF-R2. These results suggest that TNF-R2 initiates signals for the proliferation of thymocytes and cytotoxic T cells, whereas TNF-R1 initiates signals for cytotoxicity and the induction of the protective activity, manganous superoxide dismutase. The nonredundant signaling observed for the two TNF receptors cannot be explained simply by the differential expression of the two TNF receptors in the various cell types, because LM cells express on their surface higher levels of TNF-R2 than TNF-R1, and LM cells, NIH 3T3 cells, and thymus cells all express mRNA corresponding to both receptor types. It is therefore likely that the two receptors initiate distinct signaling pathways that result in the induction of different cellular responses. (17)(18)(19)(20).A number of recent reports have described initial studies investigating the individual roles of the two human TNF receptors. Polyclonal and monoclonal antibodies directed against human TNF-R1 have been shown to behave as receptor agonists and elicit several TNF activities, such as cytotoxicity, fibroblast proliferation, resistance to chlamidiae, and synthesis of prostaglandin E2 (15,21,22). Monoclonal antibodies against human TNF-R1 that block the binding of TNF to TNF-R1 and antagonize several TNF effects have also been described (21-23). Although no direct signaling role for TNF-R2 has yet been identified with either receptor agonists or transfection studies, several reports have described monoclonal antibodies directed against TNF-R2 that can partially antagonize TNF responses (such as cytotoxicity and activation of NF-KB) and enhance the antagonistic effects of anti-TNF-R1 monoclonal antibodies (22)(23)(24). These reports suggested that both TNF receptors are active in signal transduction and that there is redundancy in the function of the two receptors. However, the reported effects of the TNF-R2 antagonists have been quite small and were observed exclusively at very low TNF concentrations. It is therefore possible that TNF-R2 is only participating as a minor accessory component to TNF-R1 ...

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The ilvB locus of Escherichia coli K-12 encloses two open reading frames defining polypeptides of 60,000 and 11,200 molecular weight. The entire locus, about 2.3 kb, is co-transcribed as an operon. The molecular weights and amino acid compositions of the presumptive operon polypeptides agree with those of the large and small subunit polypeptides of acetohydroxyacid synthase (AHAS) I, for which ilvB is the structural locus. We reserve the designation ilvB for the promoter proximal (longer) cistron and designate the promoter distal cistron ilvN. The molecular weight and amino acid sequence of the ilvB polypeptide are strikingly similar to those of the I1vI (larger subunit of AHAS III) and I1vG (larger subunit of AHAS II) polypeptides. There is less size uniformity among the I1vN, I1vH (smaller subunit of AHAS III), and I1vM (smaller subunit of AHAS II) polypeptides. Nevertheless, there is significant amino acid sequence homology among the three small subunit polypeptides. Thus, all three AHAS isozymes of E. coli K-12 probably have a common evolutionary origin.

We report here that infection of the human

An expression plasmid encoding the extracellular domain of the 75-kDa human tumor necrosis factor (TNF) type 2 receptor (TNF-R2) was constructed and used to generate a stable cell line secreting soluble TNF-R2 (sTNF-R2). Purified sTNF-R2 was resolved by SDS-PAGE into one band of approximate M(r) 43,000, consistent with a molecular weight of 36,000 +/- 4800 obtained by sedimentation equilibrium analysis. The apparent molecular weight observed by gel filtration chromatography was approximately 136,000. Glycosylation analysis revealed that Asn-149 is fully glycosylated, while Asn-171 is incompletely glycosylated (approximately 50%), and that a proline-, serine-, and threonine-rich region (residues 175-234) contains O-linked carbohydrate structures. Scatchard analysis of [125I]TNF-alpha and [125I]TNF-beta binding to sTNF-R2 gave dissociation constants (Kd) of 0.3 and 0.75 nM, respectively, comparable to those observed for intact cell-surface TNF-R2. The sTNF-R2 was found to block the cytotoxicity of both TNF-alpha and TNF-beta in a murine L-M cell assay. The sizes of the sTNF-R2.TNF-alpha and sTNF-R2.TNF-beta complexes determined by gel filtration chromatography were approximately 322 and 204 kDa, respectively. The stoichiometry of the sTNF-R2.TNF-alpha and sTNF-R2.TNF-beta complexes were examined by size-exclusion chromatography, sedimentation equilibrium, and cross-linking. The data from these studies suggest that at least two molecules of sTNF-R2 can bind to a single TNF-alpha or TNF-beta trimer.