Abstract
The p55 and p75 tumor necrosis factor receptors are known to mediate their effects on cells through distinct signaling pathways. Under certain circumstances, the two classes of TNF receptors cooperate with each another to produce enhanced cellular responses. The only molecular mechanism proposed thus far to explain this effect is the process of "ligand passing," whereby TNF is concentrated at cell surfaces by binding to p75 and then following dissociation from this receptor class binds with high efficiency to p55. Using the in vivo model of TNF-induced TNF receptor shedding we have uncovered a novel ligand-dependent interaction of the two TNF receptors that occurs upon exposure of cells to TNF. Using TNF receptor-specific monoclonal antibodies that bind TNF receptors in the presence or absence of ligand, we report that TNF induces the formation of heterocomplexes consisting of both p55 and p75 TNF receptors. Whereas immunoprecipitates from untreated or human TNF-treated cells formed with either p55 or p75 TNF receptor-specific monoclonal antibodies contained only the relevant TNF receptor class, anti-p55 or anti-p75 precipitated both receptor types from murine TNF-treated cells. Ligand-induced complex formation was transient, occurred at physiologically relevant concentrations of TNF, and occurred with receptors lacking intracellular domains or that contained irrelevant transmembrane domains. Formation of TNF receptor heterocomplexes may therefore 1) define a novel molecular mechanism of ligand passing and/or 2) contribute to cooperative TNF receptor signaling via the juxtaposition of the intracellular domains of the two receptor classes and the signaling proteins that they recruit.
Highlights
TNF1 interacts with two distinct receptors of Mr 55,000 and 75,000, which are independently expressed on cell surfaces [1, 2]
Maximal TNF-induced p75 Shedding Requires Engagement of Both p55 and p75—It is well established that TNF can effect shedding of the p75 TNF receptor in vivo [19]
To investigate whether p55 ligation was necessary for TNFmediated p75 shedding, we examined whether murine TNF effected the shedding response under circumstances where p55 ligation could not occur
Summary
Reagents and Materials—Monoclonal antibodies specific for murine TNF␣ [22] and murine p55 and p75 [18] were produced as described and biotinylated using the Enzotin reagent (ENZO Biochem, Inc., New York, NY) according to the manufacturer’s protocol. In Vivo TNF Receptor Shedding Experiments—Mice were injected intraperitoneally with 0.5-ml samples of monoclonal antibodies or pyrogen-free physiologic saline. At various time points thereafter lipopolysaccharide or purified recombinant murine or human TNF was diluted in 0.5 ml of pyrogen-free saline and was injected intraperitoneally into the mice. At specified time points mice were bled and the serum was immediately assayed for the presence of shed TNF receptor proteins by enzyme-linked immunosorbent assay. The total number of TNF binding sites was assessed by offering 0.5, 1, 2, 4, 6, 8, 10, 15, 25, 50, 75, 100, or 125 ng of radiolabeled MuTNF to 4 million Meth A cells for 1 h at 4 °C. Nuclear extracts were prepared as described [23] and NF-B activation quantitated using an electrophoretic mobility shift assay that employed a 32P-labeled 27-base pair oligonucleotide probe derived from the promoter region of the Ig gene [24]
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