Interleukin-36 (IL-36) ligands require processing for full agonist (IL-36α, IL-36β, and IL-36γ) or antagonist (IL-36Ra) activity

IL-36α, IL-36β, and IL-36γ (formerly IL-1F6, IL-1F8, and IL-1F9) are IL-1 family members that signal through the IL-1 receptor family members IL-1Rrp2 (IL-1RL2) and IL-1RAcP. IL-36RA (formerly IL-1F5) has been reported to antagonize IL-36γ. However, our previous attempts to demonstrate IL-36RA antagonism were unsuccessful. Here, we demonstrate that IL-36RA antagonist activity is dependent upon removal of its N-terminal methionine. IL-36RA starting at Val-2 is fully active and capable of inhibiting not only IL-36γ but also IL-36α and IL-36β. Val-2 of IL-36RA lies 9 Amino acids N-terminal to an A-X-Asp motif conserved in all IL-1 family members. In further experiments, we show that truncation of IL-36α, IL-36β, and IL-36γ to this same point increased their specific activity by ∼10(3)-10(4)-fold (from EC(50) 1 μg/ml to EC(50) 1 ng/ml). Inhibition of truncated IL-36β activity required ∼10(2)-10(3)-fold excess IL-36RA, similar to the ratio required for IL-1RA to inhibit IL-1β. Chimeric receptor experiments demonstrated that the extracellular (but not cytoplasmic) domain of IL-1Rrp2 or IL-1R1 is required for inhibition by their respective natural antagonists. IL-36RA bound to IL-1Rrp2, and pretreatment of IL-1Rrp2-expressing cells with IL-36RA prevented IL-36β-mediated co-immunoprecipitation of IL-1Rrp2 with IL-1RAcP. Taken together, these results suggest that the mechanism of IL-36RA antagonism is analogous to that of IL-1RA, such that IL-36RA binds to IL-1Rrp2 and prevents IL-1RAcP recruitment and the formation of a functional signaling complex. In addition, truncation of IL-36α, IL-36β, and IL-36γ dramatically enhances their activity, suggesting that post-translational processing is required for full activity.