title: Interleukin-36 (IL-36) ligands require processing for full agonist (IL-36α, IL-36β, and IL-36γ) or antagonist (IL-36Ra) activity.
abstract: 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.
authors.affiliation: Department of Inflammation Research, Amgen Incorporated, Seattle, Washington 98119. Electronic address: townej@amgen.com.
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Towne
authors.affiliation: Department of Inflammation Research, Amgen Incorporated, Seattle, Washington 98119.
authors.forename: Blair R
authors.initials: BR
authors.lastname: Renshaw
authors.affiliation: Department of Inflammation Research, Amgen Incorporated, Seattle, Washington 98119.
authors.forename: Jason
authors.initials: J
authors.lastname: Douangpanya
authors.affiliation: Department of Inflammation Research, Amgen Incorporated, Seattle, Washington 98119.
authors.forename: Brian P
authors.initials: BP
authors.lastname: Lipsky
authors.affiliation: Department of Protein Sciences, Amgen Incorporated, Seattle, Washington 98119.
authors.forename: Min
authors.initials: M
authors.lastname: Shen
authors.affiliation: Department of Inflammation Research, Amgen Incorporated, Seattle, Washington 98119.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Gabel
authors.affiliation: Department of Inflammation Research, Amgen Incorporated, Seattle, Washington 98119.
authors.forename: John E
authors.initials: JE
authors.lastname: Sims
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D007375:Interleukin-1 / Q000819:agonists* / Q000037:antagonists & inhibitors* / Q000378:metabolism; D007378:Interleukins / Q000737:chemistry / Q000378:metabolism*; D019169:Jurkat Cells; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D017472:Receptors, Interleukin-1 / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D007375:Interleukin-1; D007378:Interleukins; D008024:Ligands; D017472:Receptors, Interleukin-1; C502352:IL-1F8 protein, mouse; C569167:IL1F9 protein, mouse; C508586:IL36A protein, human; C508588:IL36B protein, human; C508589:IL36G protein, human; C122187:IL36RN protein, human; C543716:interleukin 1F6, mouse; C569500:Il1rl2 protein, mouse