Temperature-sensitive Differential Affinity of TRAIL for Its Receptors

Abstract

TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines which induces apoptotic cell death in a variety of tumor cell lines. It mediates its apoptotic effects through one of two receptors, DR4 and DR5, which are members of of the TNF receptor family, and whose cytoplasmic regions contain death domains. In addition, TRAIL also binds to 3 "decoy" receptors, DcR2, a receptor with a truncated death domain, DcR1, a glycosylphosphatidylinositol-anchored receptor, and OPG a secreted protein which is also known to bind to another member of the TNF family, RANKL. However, although apoptosis depends on the expression of one or both of the death domain containing receptors DR4 and/or DR5, resistance to TRAIL-induced apoptosis does not correlate with the expression of the "decoy" receptors. Previously, TRAIL has been described to bind to all its receptors with equivalent high affinities. In the present work, we show, by isothermal titration calorimetry and competitive enzyme-linked immunosorbent assay, that the rank order of affinities of TRAIL for the recombinant soluble forms of its receptors is strongly temperature dependent. Although DR4, DR5, DcR1, and OPG show similar affinities for TRAIL at 4°C, their rankordered affinities are substantially different at 37°C, with DR5 having the highest affinity (K D < 2 nM) and OPG having the weakest (K D ‫؍‬ 400 nM). Preferentially enhanced binding of TRAIL to DR5 was also observed at the cell surface. These results reveal that the rank ordering of affinities for protein-protein interactions in general can be a strong function of temperature, and indicate that sizeable, but hitherto unobserved, TRAIL affinity differences exist at physiological temperature, and should be taken into account in order to understand the complex physiological and/or pathological roles of TRAIL.

Homomeric and heteromeric interactions of the extracellular domains of death receptors and death decoy receptors

“…Particularly, the measured affinity of sTRAIL with sDR5 was equivalent with the previously reported value for E. coli-produced sDR5 ($16 nM) as determined by SPR [13]. The affinity rank of sTRAIL for the soluble receptors of sDR5 > sDR4 > sDcR1 > and DcR2 was consistent with previous results for sDRs-Fc and sDcRs-Fc [24]. The slightly higher affinity of sTRAIL with sDR5 is mainly attributed to 3-to 7-fold faster k on with similar k off , as compared to other receptors (Table 1).…”

“…The estimated K D of the interaction of sTRAIL with sDR5 was approximately 10 nM, which was slightly stronger than sDR4 ($29 nM), sDcR1 ($47 nM), and sDcR2 ($62 nM) ( Table 1). The K D values were within the ranges of those reported for mammalian cell-produced immunoglobulin Fc-fused sDRs (sDRs-Fc) and sDcRs (sDcRs-Fc), in which affinities for sTRAIL were $0.5-59 nM for sDR5-Fc, $0.5-70 nM for sDR4-Fc, $1-200 nM for sDcR1-Fc, and $39 nM for sDcR2-Fc as measured by isothermal titration calorimetry [24], isotope-labeled equilibrium binding assay [8], and SPR [20,21]. Particularly, the measured affinity of sTRAIL with sDR5 was equivalent with the previously reported value for E. coli-produced sDR5 ($16 nM) as determined by SPR [13].…”

Differential Regulation of the Expression of CD95 Ligand, Receptor Activator of Nuclear Factor-κB Ligand (RANKL), TNF-Related Apoptosis-Inducing Ligand (TRAIL), and TNF-α During T Cell Activation

“…To investigate whether the activity is due to activation-induced production of TRAIL, we employed soluble TRAIL receptor DR5 (69) and a decoy receptor OPG. DR5 is specific to TRAIL, and OPG blocks the activity of both TRAIL and RANKL (70). We found that both fusion proteins effectively blocked the cytotoxic activity of supernatant from activated A1.1 cells, indicating that the cytotoxic activity of the supernatant from activated A1.1 cells is due to the presence of TRAIL.…”

FLICE-Like Inhibitory Protein Blocks Transforming Growth Factor β1–Induced Caspase Activation and Apoptosis in Prostate Epithelial Cells

“…Specifically, because TRAIL kills neoplastic and normal human prostate epithelial cells (18) via activation of the apical caspases (43), we examined its role in TGFh1-mediated apoptosis. Human TRAIL can bind two death-inducing receptors, DR4 and DR5, as well as three decoy receptors and OPG (44), but the DR4 receptor does not exist in rodents (45). Fc fusions of the extracellular domains of these receptors block TRAIL-induced cell death (18).…”