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 degrees C, their rank-ordered affinities are substantially different at 37 degrees 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.