Identification of Essential Regions in the Cytoplasmic Tail of Interleukin-1 Receptor Accessory Protein Critical for Interleukin-1 Signaling

Abstract

Interleukin-1 (IL-1) 1 possesses a wide spectrum of inflammatory, metabolic, physiological, hematopoetic, and immunological properties. IL-1 binds to two different receptors of the Ig superfamily: IL-1 receptor type I (IL-1RI) and IL-1 receptor type II (IL-1RII). Both receptors bind the ligands with distinct affinities (1). Whereas IL-1RI is necessary for signal transduction (2), IL-1RII is not capable of transducing an activation signal but rather acts as a decoy receptor serving as a ligand sink and competing for IL-1 with the IL-1RI (3-5). An additional member of the IL-1 receptor family, IL-1 receptor accessory protein (IL-1RAcP), has also been identified. The 66-kDa IL-1RAcP shares limited homology with IL-1RI and IL-1RII but does not bind IL-1 (6). It has been shown that co-expression of the IL-1RAcP is essential for a fully functional IL-1RI complex (7-10).In the last years the elucidation of early IL-1 signaling events has progressed. After ligand-induced complex formation of IL-1RI and IL-1RAcP (7-11), the Ser/Thr kinase IRAK is recruited to the receptor complex, where it becomes highly phosphorylated (12, 13). Recruitment of IRAK requires the intracellular domains of both receptor chains (8,10,14). Dominant-negative forms of MyD88 block IL-1 signaling (15). After phosphorylation by itself (12) or by additional kinases (16), IRAK leaves the receptor complex and interacts with tumor necrosis factor receptor-associated factor 6 (17). Recently, the MAP kinase kinase kinase TAK1 has been identified to interact with tumor necrosis factor receptor-associated factor 6 in association with , thereby providing a link to the machinery that activates nuclear factor B (NF-B) via stimulation of IB kinases (IKK␣ and IKK␤) (23-28). One critical component in the signaling cascade is MyD88 (29, 30), because mice lacking this adaptor molecule do not show cytokine-induced activation of NF-B and c-Jun N-terminal kinase (JNK) (31).We have previously described an IL-1RI-positive subclone of EL4 cells, EL4D6/76, which binds IL-1 with high affinity but fails to activate NF-B or produce IL-2 following IL-1 stimulation (7,32). This defect is due to the lack of IL-1RAcP expression and can be overcome by transfection with IL-1RAcP, thus reconstituting IL-1-specific functional defects in EL4D6/76 cells (7,9). In the present study we investigated regions within the cytoplasmic domain of IL-1RAcP that are required for perpetuating IL-1 responses. We present data demonstrating that, in addition to box 3 (aa 538 -542) of the TIR domain, aa 527-534 within the cytoplasmic domain of IL-1RAcP are essential for IL-1 signaling. EXPERIMENTAL PROCEDURES Construction of Truncated and Mutated Forms of IL-1RAcP-Trun-cated forms of IL-1RAcP were constructed by PCR technique. The vector pEF-IL-1RAcP containing the murine IL-1RAcP coding sequence was used as a template (9). Truncated fragments were cloned into pFLAG-IL-1RAcP (kind gift of Michael Martin, Hannover, Germany), a *

Soluble interleukin‐1 receptor accessory protein ameliorates collagen‐induced arthritis by a different mode of action from that of interleukin‐1 receptor antagonist

“…Membrane IL-1RAcP is a crucial component of the IL-1 signaling complex, stabilizing the IL-1-IL-1RI complex and mediating IL-1 signaling (7,16,17,36). The recently discovered naturally occurring alternative splice transcript of the membrane IL-1RAcP comprises the 3 extracellular Ig-like domains (4,22,23).…”

“…Another member of the IL-1 receptor family is IL-1RII (68 kd) (15), which upon binding of IL-1 also associates with IL-1RAcP. However, this does not lead to signal transduction because this receptor lacks the intracellular Toll-IL-1 receptor domain that is crucial for MyD88 binding (16)(17)(18). Therefore, the type II receptor is a decoy receptor as has been described for its transmembrane and soluble forms (19).…”

The Interleukin 1 (IL-1) Receptor Accessory Protein Toll/IL-1 Receptor Domain

“…Briefly, the transfection reagent was freshly prepared by mixing 300 l of 0.5 mg/ml DEAE-dextran in TBS (25 mM Tris-HCl, pH 7.4, 123 mM NaCl, 5 mM KCl, 0.7 mM CaCl 2 , 0.5 mM MgCl 2 , 0.6 mM Na 2 HPO 4 ) and 300 l of chloroquine (80 g/ml in TBS) containing 500 ng of IL-1RAcP expression plasmid encoding the various mutated forms of the coreceptor chain together with 1 g of a luciferase reporter plasmid containing the IL-1-inducible proximal region of the murine IL-2 promoter (pGL3-IL-2(-303)) (38,39). 5 ϫ 10 6 cells (in logarithmic growth phase) were washed in phosphate-buffered saline, pH 7.4 and resuspended in TBS.…”

“…IL-2 promotor activation was determined by measuring the luciferase activity in transiently transfected cells using the LucLite Plus luciferase reporter gene assay kit (PerkinElmer Life Sciences) as described previously (39). Luciferase activity was normalized to the IL-1R complex-independent IL-18 response obtained after co-incubation of the transfectants in the presence of PMA ϩ IL-18.…”

IL-1 receptor accessory protein is essential for IL-33-induced activation of T lymphocytes and mast cells

“…Both transmembrane proteins form a heterodimer, which results in the close association of the cytosolic Toll-like IL-1R (TIR) homology domains (3). IL-1RAcP is essential for IL-1 signaling (4)(5)(6), and the TIR domains of both chains are required to facilitate signaling (7,8). Ligand-mediated heterodimerization with subsequent intracellular association of strongly homologous, but discrete, TIR domains is a typical feature of the IL-1 receptor subfamily of TIR-domain-containing receptors.…”