Parallel experiments demonstrated that substitution of the membrane-proximal residue Asp448, with predicted effects on -helical, turn, and coil regions of the protein, significantly reduced formation of the TILRR-containing IL-1RI complex, in agreement with a relevance of signaling receptor association to TILRR function (Fig

Parallel experiments demonstrated that substitution of the membrane-proximal residue Asp448, with predicted effects on -helical, turn, and coil regions of the protein, significantly reduced formation of the TILRR-containing IL-1RI complex, in agreement with a relevance of signaling receptor association to TILRR function (Fig. enhance recruitment of MyD88 and control Ras-dependent amplification of NF-B and inflammatory responses. Keywords:Cytoskeleton/Actin, Proteoglycans Structure, Receptors/Cytokine, Receptors/Structure-Function, Signal Transduction/Adapter Proteins, Interleukin-1 Receptor Type 1, NF-kappaB Signaling, Receptor Complex Formation and Bufalin Stoichiometry == Introduction == Immune and inflammatory response are regulated by Toll-like and IL-12receptors (14). Their activation is initiated by ligand binding and co-receptor association to the receptor complex, followed by recruitment of distinct Spry3 sets of adapter proteins to the cytoplasmic Toll/IL-1 receptor domain, common to all members of the family. The IL-1 receptor subfamily is a continuously growing group of regulatory proteins, named after the initially identified type I signaling receptor (IL-1RI) (5,6). Its activation is regulated by the proinflammatory cytokines IL-1 and IL-1 and the receptor antagonist, IL-1ra, and by association of the IL-1R accessory protein (AcP) with the receptor complex (6). IL-1 responses are, in addition, linked to events related to cell attachment and the cytoskeleton and controlled by the small GTPases (7,8). Receptor activation, followed by recruitment of the MyD88 adapter and by association of the receptor-associated kinases (IRAKs) and TRAF6, leads to induction of the mitogen-activated protein kinase cascade and the NF-B pathway (13,9,10). The importance of receptor complex composition and stoichiometry in adapter protein recruitment and TIR signaling is well recognized (4,1113). Our earlier studies have identified a cell surface heparan sulfate proteoglycan, which is recruited to the IL-1RI complex in a substrate-dependent manner (14,15). Here we report on the cloning and characterization of this novel co-receptor, TILRR (Toll-like/IL-1 receptor regulator). We show that TILRR associates with the extracellular domain of IL-1RI and magnifies NF-B-regulated inflammatory responses by potentiating recruitment of MyD88 and linking TIR activation with mechanotransduction by controlling induction of the Ras GTPase. == EXPERIMENTAL PROCEDURES == == == == == == Tissue Culture == Murine and human cell lines and primary cells were cultured under conventional conditions. HeLa cells, endothelial cells (HMEC-1, CDC, Ades, Lawley, and Candal), fibroblasts (NIH 3T3 and primary human gingival fibroblasts, HGF), monocytes (THP1, J774, BL413, ML-1, HL-60, U937, MOLT4, and K562), macrophages (Raw 264.7), embryonic kidney (HEK293), mouse epithelial cells (IL-1RI-transformed Bufalin C127 cells), and primary human peripheral blood mononuclear cells (PBMCs) were cultured at 37 C and 5% CO2in DMEM (Invitrogen) with 10% fetal calf serum (Invitrogen or Cambrex), penicillin (50 units/ml), and streptomycin (50 g/ml) (Invitrogen) or in MCDB 131 (Invitrogen) with 10% fetal calf serum, epidermal growth factor (10 ng/ml), hydrocortisone (1 g/ml), andl-glutamine (2 mm) (all from Bufalin Sigma) and the C127, in addition, with G418 (500 g/ml). Cells were detached in EDTA (5 mm, Sigma) and plated on bare plastic or on fibronectin (10 g/ml; Sigma) to enhance levels of endogenous TILRR (luciferase assays, 96-well plates (6.0 103cells/well); confocal microscopy, 8-well chamber slides (12.0 103cells/well); Western analysis, 24-well plates (35 103cells/well); enzyme-linked immunosorbent assays, 6-well plates (2 105cells/well); radioreceptor assays and immunoprecipitation, 10-cm dishes (1.0 106cells)) and stimulated with IL-1 (1 nm) (a kind gift of Dr. Steve Pool (National Institute for Biological Standards and Control)), TNF (10 ng/ml) (Promega), PDGFBB (25 ng/ml) (R&D Systems), or TGF-2 (3 ng/ml) (R&D Systems) 2448 h after transfection. == Protein Identification == In-gel digestion and MALDI-TOF was carried out as previously (16), and data were analyzed by Profound, MASCOT, MOWSE, and PeptIdent. Sequence analysis and alignments used.