The exon-3-encoded domain of IL-15Rα contributes to IL-15 high-affinity binding and is crucial for the IL-15 antagonistic effect of soluble IL-15Rα

We previously showed that a natural soluble form of interleukin-15 (IL-15) R alpha corresponding to the full-length ectodomain of IL-15R alpha behaved as a potent antagonist of IL-15 action through whereas a recombinant soluble IL-15R alpha sushi domain did not, but instead acted as all agonist of IL-15 action through IL-15R beta/gamma. In order to determine precisely the molecular basis governing these antagonistic versus agonistic actions, we compared the binding properties and biological effects of recombinant Soluble IL-15R alpha (sIL-15R alpha) species containing the sushi domain and different remaining parts of the ectodomain. We first demonstrate that the exon-3-encoded domain and, more particularly, its N-terminal 13-amino-acid (aa) peptide are important, in addition to the adjacent exon-2-encoded sushi domain, for the stabilization of the high-affinity IL-15.IL-15R alpha complex by slowing down its dissociation rate and by contributing to about 10-20% of the free energy of interaction. We next show that all sushi-containing sIL-15R alpha are agonists on IL-15R beta/gamma, coordinately increasing IL-15 binding and IL-15-induced proliferation. Their agonistic potencies are proportional to their respective affinities for IL-15. We then show that the antagonistic effect of sIL-15R alpha in the context of IL-15R alpha/beta/gamma is due to the 1.3-aa peptide that creates a sterical constraint impeding the binding of the sIL-15R alpha.IL-15 complex to the membrane-anchored IL-15R alpha/beta/gamma. In the frame of the soluble IL-15R alpha sushi domain-IL-15 fusion protein that contains the 13-aa peptide, this constraint is alleviated as a result of a conformational effect due to the covalent]inking of the 13-aa peptide to the N-terminus of IL-15. The soluble IL-15Ra sushi domain-IL-15 fusion protein is therefore able to bind and activate both the IL-15R beta/gamma and the IL-15R alpha/beta/gamma receptors. (C) 2008 Elsevier Ltd. All rights reserved.