Studies of Binding of Tumor Necrosis Factor (TNF)-like Weak Inducer of Apoptosis (TWEAK) to Fibroblast Growth Factor Inducible 14 (Fn14)

To perform highly sensitive cellular binding studies with TNF-like weak inducer of apoptosis (TWEAK), we developed a bioluminescent variant of soluble TWEAK (GpL-FLAG-TNC-TWEAK) by fusing it genetically to the C terminus of the luciferase of Gaussia princeps (GpL). Equilibrium binding studies on human (HT1080 and HT29) and murine (Renca and B16) cell lines at 37 degrees C revealed high affinities of human TWEAK from 53 to 112 pM. The dissociation rate constant of the TWEAK-Fn14 interaction was between 0.48 x 10(-3) s(-1) (HT29) and 0.58 x 10(-3) s(-1) (HT1080) for the human molecules, and the association rate constant obtained was 3.3 x 10(6) M-1 s(-1) for both cell lines. It has been shown previously that oligomerization of soluble TWEAK trimers results in enhanced Fn14-mediated activation of the classical NF kappa B pathway. Binding studies with GpL-FLAG-TNC-TWEAK trimers oligomerized by help of a FLAG tag-specific antibody gave no evidence for a major increase in Fn14 occupancy by oligomerized ligand despite strongly enhanced induction of the NF kappa B target IL8. Thus, aggregated complexes of soluble TWEAK and Fn14 have a higher intrinsic activity to stimulate the classical NF kappa B pathway and qualitatively differ from isolated trimeric TWEAK-Fn14 complexes. Furthermore, determination of IL8 induction as a function of occupied activated receptors revealed that the intrinsic capability of TNFR1 to stimulate the classical NF kappa B pathway and IL8 production was similar to 100-fold higher than Fn14. Thus, although similar to 25 activated TNFR1 trimers were sufficient to trigger half-maximal IL8 production, more than 2500 cell-bound oligomerized TWEAK trimers were required to elicit a similar response.