The binding of heterotrimeric lymphotoxin LTα1β2 towards the LTβ receptor (LTβR) a member of the tumor necrosis factor receptor (TNFR) superfamily induces nuclear factor κB (NF-κB) activation and cell death in HT29 adenocarcinoma cells. The mutant and endogenous TRAF3s are both recruited at equimolar amounts to the LTβR suggesting that this mutant disrupts the function of the signaling complex. These results implicate TRAF3 as a critical component of the LTβR death signaling complex and indicate that at least two impartial signaling pathways are initiated Amprenavir by LTβR ligation. translation. Immune complexes were detected with donkey anti-rabbit IgG coupled to Amprenavir horseradish peroxidase and chemiluminescence substrate (ECL reagent; Amersham) with a 15-min exposure. The monoclonal antibodies used were anti-LTβR BDA8 [mouse IgG1 (10) a gift from J. Browning]; anti-Fas CH11 (mouse IgM; MBL Nagoya Japan); anti-TNFR60 H398 (mouse IgG2a Biosource Camarillo CA); and antibodies to intracellular adhesion molecule 1 (ICAM-1) (mouse IgG1 Chemicon Temecula CA). TRAF3 Mutant and Transfection. The TRAF3 deletion mutant encoding amino acids 368-568 was designed by PCR amplification (DNA polymerase) from TRAF3 cDNA using the following oligonucleotides: 5′ primer 5′-CCGGATCCATGGACTACAAGGACGACGATGACAAGAGCGCGGGGCAAGTG-3′ which introduces a < 0.002; Table ?Table1).1). The initial pool of G418-resistant TRAF3Δ1-367-transfected cells also experienced an attenuated response to LTα1β2 (IC50 = 2000 pM; data not shown) indicating that the eight clones are not rare in the initial population. However the TRAF3Δ1-367-expressing clones were similar to the control lines in sensitivity to Fas antibody-induced apoptosis (Fig. ?(Fig.22 and Table ?Table1).1). Interestingly the TRAF3Δ1-367 expressing clones were somewhat attenuated in their sensitivity to TNF-induced cell death as compared with the control lines (= 0.03; Table ?Table1).1). Thus TRAF3Δ1-367 inhibits LTβR-ligand-induced cell death has no effect on Fas-induced cell death and appears to have a small effect on TNF-induced cell death. Physique 2 A TRAF3 mutant inhibits cell death by LTβR. The HT29.14S clones expressing TRAF3Δ1-367 were incubated in medium containing either recombinant cytokines (soluble LTα1β2 or TNF) or receptor-specific antibodies (purified ... Table 1 Effect of TRAF3Δ1-367 on ligand-induced cell death N-Terminally Truncated TRAF3 Does Not Inhibit LTβR-Ligand-Induced NF-κB Activation. Two clones which express TRAF3Δ1-367 and are highly resistant to LTβR-ligand-induced cell death were compared with the pool of control vector-transfected cells for LTβR-ligand-induced NF-κB activation. The TRAF3Δ1-367-expressing clones did not differ from control vector-expressing cells in surface LTβR Fas or TNFR60 expression as measured by circulation cytometry (data not shown). Activation of TRAF3Δ1-367-expressing or control HT29.14Svec cells for 15 min with LTα1β2 or antibodies to LTβR specifically induced comparable levels of NF-κB activation as revealed by an electrophoretic mobility-shift assay (Fig. ?(Fig.33A). TNF was also similarly efficient at inducing activation of NF-κB in the TRAF3Δ1-367 expressing and control HT29.14Svec cells (Fig. ?(Fig.33A). Anti-Fas monoclonal antibody CH11 induced NF-κB poorly although it is usually a very potent transmission transducer for apoptosis in these cells which is usually consistent with apoptosis and NF-κB activation being individual pathways in these cells. Antibodies to the p65 or p50 subunits of NF-κB but not to c-Rel Rel B or p52 super-shifted the κB oligonucleotide indicating that LTα1β2 activates a p65·p50 heterocomplex much like TNF (Fig. ?(Fig.33B). The expression of ICAM-1 an adhesion molecule regulated in part by NF-κB (55) is usually modestly enhanced on HT29.14S cells by LTα1β2 or TNF with a shift Amprenavir in mean peak fluorescence of 50-80% 14 hr after activation. TRAF3Δ1-367-expressing and control HT29.14Svec cells did not differ in LTα1β2-induced ICAM-1 expression (not shown). These results indicate that TRAF3Δ1-367 expression does not impact WAS LTβR-ligand-induced NF-κB activation or ICAM-1 expression. Physique Amprenavir 3 Activation of NF-κB by LTβR. (A) HT29.14S clones 7 and 8 transfected with TRAF3Δ1-367 mutant or empty pCDNA3 vector (V) were treated for 15 min with normal goat IgG (10 μg/ml) (lanes 1-3) LTα … TRAF3Δ1-367 Expression Does Not Inhibit TRAF3 Recruitment to LTβR. To investigate whether TRAF3Δ1-367 interferes with LTβR signaling by binding to the LTβR and preventing endogenous wild-type TRAF3 recruitment ligand-dependent recruitment of TRAF3 and.