Temperature-sensitive differential affinity of TRAIL for its receptors - DR5 is the highest affinity receptor

TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines wh ich induces apoptotic cell death in a variety of tumor cell lines. It media tes its apoptotic effects through one of two receptors, DR4 and DR5, which are members of of the TNF receptor family, and whose cytoplasmic regions co ntain death domains. In addition, TRAIL also binds to 3 "decoy" receptors, DcR2, a receptor with a truncated death domain, DcR1, a glycosylphosphatidy linositol-anchored receptor, and OPG a secreted protein which is also known to bind to another member of the TNF family, RANKL. However, although apop tosis depends on the expression of one or both of the death domain containi ng 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 affini ties. In the present work, we show, by isothermal titration calorimetry and competitive enzyme-linked immunosorbent assay, that the rank order of affi nities of TRAIL for the recombinant soluble forms of its receptors is stron gly temperature dependent. Although DR4, DR5, DcR1, and OPG, show similar a ffinities for TRAIL at 4 degrees C, their rank-ordered affinities are subst antially different at 37 degrees C, with DR5 having the highest affinity (K -D less than or equal to 2 nM) and OPG having the weakest (K-D = 400 nM). P referentially enhanced binding of TRAIL to DR5 was also observed at the cel l surface. These results reveal that the rank ordering of affinities for pr otein-protein interactions in general can be a strong function of temperatu re, and indicate that sizeable, but hitherto unobserved, TRAIL affinity dif ferences exist at physiological temperature, and should be taken into accou nt in order to understand the complex physiological and/or pathological rol es of TRAIL.