Abstract
15 min readHost defense against infection is induced by Toll-like and interleukin (IL)-1 receptors, and controlled by the transcription factor NF-κB. Our earlier studies have shown that IL-1 activation impacts cytoskeletal structure and that IL-1 receptor (IL-1RI) function is substrate-dependent. Here we identify a novel regulatory component, TILRR, which amplifies activation of IL-1RI and coordinates IL-1-induced control with mechanotransduction. We show that TILRR is a highly conserved and widely expressed enhancer of IL-1-regulated inflammatory responses and, further, that it is a membrane-bound glycosylated protein with sequence homology to members of the FRAS-1 family. We demonstrate that TILRR is recruited to the IL-1 receptor complex and magnifies signal amplification by increasing receptor expression and ligand binding. In addition, we show that the consequent potentiation of NF-κB is controlled through IL-1RI-associated signaling components in coordination with activation of the Ras GTPase. Using mutagenesis, we demonstrate that TILRR function is dependent on association with its signaling partner and, further, that formation of the TILRR-containing IL-1RI complex imparts enhanced association of the MyD88 adapter during ligand-induced activation of NF-κB. We conclude that TILRR is an IL-1RI co-receptor, which associates with the signaling receptor complex to enhance recruitment of MyD88 and control Ras-dependent amplification of NF-κB and inflammatory responses. Host defense against infection is induced by Toll-like and interleukin (IL)-1 receptors, and controlled by the transcription factor NF-κB. Our earlier studies have shown that IL-1 activation impacts cytoskeletal structure and that IL-1 receptor (IL-1RI) function is substrate-dependent. Here we identify a novel regulatory component, TILRR, which amplifies activation of IL-1RI and coordinates IL-1-induced control with mechanotransduction. We show that TILRR is a highly conserved and widely expressed enhancer of IL-1-regulated inflammatory responses and, further, that it is a membrane-bound glycosylated protein with sequence homology to members of the FRAS-1 family. We demonstrate that TILRR is recruited to the IL-1 receptor complex and magnifies signal amplification by increasing receptor expression and ligand binding. In addition, we show that the consequent potentiation of NF-κB is controlled through IL-1RI-associated signaling components in coordination with activation of the Ras GTPase. Using mutagenesis, we demonstrate that TILRR function is dependent on association with its signaling partner and, further, that formation of the TILRR-containing IL-1RI complex imparts enhanced association of the MyD88 adapter during ligand-induced activation of NF-κB. We conclude that TILRR is an IL-1RI co-receptor, which associates with the signaling receptor complex to enhance recruitment of MyD88 and control Ras-dependent amplification of NF-κB and inflammatory responses. IntroductionImmune and inflammatory response are regulated by Toll-like and IL-1 2The abbreviations used are: ILinterleukinTNFtumor necrosis factorTGFtransforming growth factorMALDI-TOFmatrix-assisted laser desorption/ionization time-of-flightsiRNAsmall interfering RNARTreverse transcriptionEGFPenhanced green fluorescent proteinPBMCperipheral blood mononuclear cell. receptors (1.Akira S. Takeda K. Nat. Rev. Immunol. 2004; 4: 499-511Crossref PubMed Scopus (6586) Google Scholar, 2.Beutler B. Nature. 2004; 430: 257-263Crossref PubMed Scopus (1247) Google Scholar, 3.O'Neill L.A. Curr. Opin. Immunol. 2006; 18: 3-9Crossref PubMed Scopus (516) Google Scholar, 4.Gay N.J. Gangloff M. Annu. Rev. Biochem. 2007; 76: 141-165Crossref PubMed Scopus (505) Google Scholar). Their activation is initiated by ligand binding and co-receptor association to the receptor complex, followed by recruitment of distinct 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.Dinarello C.A. J. Exp. Med. 2005; 201: 1355-1359Crossref PubMed Scopus (402) Google Scholar, 6.Sims J.E. Curr. Opin. Immunol. 2002; 14: 117-122Crossref PubMed Scopus (179) Google Scholar). Its activation is regulated by the proinflammatory cytokines IL-1α and IL-1β and the receptor antagonist, IL-1ra, and by association of the IL-1R accessory protein (AcP) with the receptor complex (6.Sims J.E. Curr. Opin. Immunol. 2002; 14: 117-122Crossref PubMed Scopus (179) Google Scholar). IL-1 responses are, in addition, linked to events related to cell attachment and the cytoskeleton and controlled by the small GTPases (7.Qwarnström E.E. MacFarlane S.A. Page R.C. Dower S.K. Proc. Natl. Acad. Sci. U.S.A. 1991; 88: 1232-1236Crossref PubMed Scopus (69) Google Scholar, 8.Caunt C.J. Kiss-Toth E. Carlotti F. Chapman R. Qwarnstrom E.E. J. Biol. Chem. 2001; 276: 6280-6288Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). 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-κB pathway (1.Akira S. Takeda K. Nat. Rev. Immunol. 2004; 4: 499-511Crossref PubMed Scopus (6586) Google Scholar, 2.Beutler B. Nature. 2004; 430: 257-263Crossref PubMed Scopus (1247) Google Scholar, 3.O'Neill L.A. Curr. Opin. Immunol. 2006; 18: 3-9Crossref PubMed Scopus (516) Google Scholar, 9.Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (974) Google Scholar, 10.Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z. Immunity. 1997; 7: 837-847Abstract Full Text Full Text PDF PubMed Scopus (915) Google Scholar).The importance of receptor complex composition and stoichiometry in adapter protein recruitment and TIR signaling is well recognized (4.Gay N.J. Gangloff M. Annu. Rev. Biochem. 2007; 76: 141-165Crossref PubMed Scopus (505) Google Scholar, 11.Gay N.J. Gangloff M. Weber A.N. Nat. Rev. Immunol. 2006; 6: 693-698Crossref PubMed Scopus (154) Google Scholar, 12.Akira S. Yamamoto M. Takeda K. Biochem. Soc. Trans. 2003; 31: 637-642Crossref PubMed Scopus (166) Google Scholar, 13.O'Neill L.A. Bowie A.G. Nat. Rev. Immunol. 2007; 7: 353-364Crossref PubMed Scopus (1971) Google Scholar). 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.Vallés S. Tsoi C. Huang W.Y. Wyllie D. Carlotti F. Askari J.A. Humphries M.J. Dower S.K. Qwarnström E.E. J. Biol. Chem. 1999; 274: 20103-20109Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 15.Valles S. Caunt C.J. Walker M.H. Qwarnstrom E.E. Lab. Invest. 2002; 82: 855-862Crossref PubMed Scopus (3) Google Scholar). 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-κB-regulated inflammatory responses by potentiating recruitment of MyD88 and linking TIR activation with mechanotransduction by controlling induction of the Ras GTPase.DISCUSSIONWe report on a novel IL-1RI co-receptor, TILRR, which associates with the signaling receptor complex to potentiate recruitment of the MyD88 adapter, control induction of the Ras GTPase, and amplify activation of NF-κB and inflammatory genes.Earlier studies identified TILRR as a heparan sulfate proteoglycan, recruited to the IL-1 receptor complex at focal adhesions (14.Vallés S. Tsoi C. Huang W.Y. Wyllie D. Carlotti F. Askari J.A. Humphries M.J. Dower S.K. Qwarnström E.E. J. Biol. Chem. 1999; 274: 20103-20109Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Involvement of TILRR in structural control is consistent with its role in induction of substrate-dependent alterations in cell shape and in activation of the Ras GTPase through IL-1RI (7.Qwarnström E.E. MacFarlane S.A. Page R.C. Dower S.K. Proc. Natl. Acad. Sci. U.S.A. 1991; 88: 1232-1236Crossref PubMed Scopus (69) Google Scholar, 8.Caunt C.J. Kiss-Toth E. Carlotti F. Chapman R. Qwarnstrom E.E. J. Biol. Chem. 2001; 276: 6280-6288Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The results thus demonstrate a link between TILRR expression and induction of Ras activity and amplification of NF-κB and suggest that TILRR functions as a coordinator of IL-1-induced responses and mechanotransduction (22.Geiger B. Spatz J.P. Bershadsky A.D. Nat. Rev. Mol. Cell Biol. 2009; 10: 21-33Crossref PubMed Scopus (1840) Google Scholar). This is further supported by its homology with members of the FRAS1 family and indicates a role for TILRR in regulation of inflammatory control in the context of tissue structure and development (21.McGregor L. Makela V. Darling. S.M. Vrontou S. Chalepakis G. Roberts C. Smart N. Rutland P. Prescott N. Hopkins J. Bentley E. Shaw A. Roberts E. Mueller R. Jadeja S. Philip N. Nelson J. Francannet C. Perez-Aytes A. Megarbane A. Kerr B. Wainwright B. Woolf A.S. Winter R.M. Scambler P.J. Nat. Genet. 2003; 34: 203-208Crossref PubMed Scopus (192) Google Scholar). Its recruitment to the receptor complex may thus regulate IL-1RI function and cellular responses in a topographical and time-dependent manner (23.Kreuger J. Spillmann D. Li J.P. Lindahl U. J. Cell Biol. 2006; 174: 323-327Crossref PubMed Scopus (395) Google Scholar).A role for TILRR in ligand binding is consistent with its lack of effect on unstimulated levels. Further, it is supported by the reduced effect of TILRR on IL-1 responses, following substitutions at residues 39 and 66, within the IL-1R ligand-binding domain (24.Vigers G.P. Anderson L.J. Caffes P. Brandhuber B.J. Nature. 1997; 386: 190-194Crossref PubMed Scopus (228) Google Scholar). It is also consistent with the reduction in binding affinity (Ka) and increase in downstream activation caused by TILRR/IL-1RI association, characteristic of cell surface glycoprotein and proteoglycan co-receptors (14.Vallés S. Tsoi C. Huang W.Y. Wyllie D. Carlotti F. Askari J.A. Humphries M.J. Dower S.K. Qwarnström E.E. J. Biol. Chem. 1999; 274: 20103-20109Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 25.Schlessinger J. Lax I. Lemmon M. Cell. 1995; 83: 357-360Abstract Full Text PDF PubMed Scopus (449) Google Scholar, 26.Fukushima K. Ikehara Y. Yamashita K. J. Biol. Chem. 2005; 280: 18056-18062Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). TILRR may thus have a role similar to that of β-glycan and gp130, which present ligand to their respective system signaling receptors (27.López-Casillas F. Wrana J.L. Massagué J. Cell. 1993; 73: 1435-1444Abstract Full Text PDF PubMed Scopus (770) Google Scholar, 28.Tanaka M. Miyajima A. Rev. Physiol. Biochem. Pharmacol. 2003; 149: 39-52Crossref PubMed Google Scholar). In addition, the increase in cell surface binding may be a consequence of the enhanced receptor expression and caused by a reduced rate of internalization, due to retention of IL-1RI at focal adhesions, following TILRR association (14.Vallés S. Tsoi C. Huang W.Y. Wyllie D. Carlotti F. Askari J.A. Humphries M.J. Dower S.K. Qwarnström E.E. J. Biol. Chem. 1999; 274: 20103-20109Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar).Effects on receptor function are expected to derive from alterations in receptor complex composition and conformation following TILRR association. With up to 70% of the receptor-bound ligand present within the high molecular weight, TILRR-containing complex, such changes are likely to have significant consequences for receptor dimerization, signal amplification, and downstream control (4.Gay N.J. Gangloff M. Annu. Rev. Biochem. 2007; 76: 141-165Crossref PubMed Scopus (505) Google Scholar, 14.Vallés S. Tsoi C. Huang W.Y. Wyllie D. Carlotti F. Askari J.A. Humphries M.J. Dower S.K. Qwarnström E.E. J. Biol. Chem. 1999; 274: 20103-20109Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Detailed analysis of effects of TILRR association on complex composition and on association of regulatory components will be carried out in future studies using crystallography, in combination with surface plasmon resonance and saturation transfer difference NMR.Sequential changes in signaling receptor conformation following TILRR association are likely to underlie the enhanced recruitment of the MyD88 adapter and the increased signal amplification (4.Gay N.J. Gangloff M. Annu. Rev. Biochem. 2007; 76: 141-165Crossref PubMed Scopus (505) Google Scholar). In addition, such changes may facilitate downstream signaling cross-talk and link TIR-induced activation with substrate-dependent control and structural regulation through the GTPases (29.Zeisel M.B. Druet V.A. Sibilia J. Klein J.P. Quesniaux V. Wachsmann D. J. Immunol. 2005; 174: 7393-7397Crossref PubMed Scopus (56) Google Scholar, 30.Kamon H. Kawabe T. Kitamura H. Lee J. Kamimura D. Kaisho T. Akira S. Iwamatsu A. Koga H. Murakami M. Hirano T. EMBO J. 2006; 25: 4108-4119Crossref PubMed Scopus (55) Google Scholar). This is consistent with the requirement of TILRR in IL-1-induced activation of the Ras GTPase and suggests that the TILRR·IL-1RI complex may constitute a platform for controlling structurally sensitive inflammatory responses by coordinating IL-1-regulated activities with mechanotransduction (31.Hahn C. Schwartz M.A. Nat. Rev. Mol. Cell Biol. 2009; 10: 53-62Crossref PubMed Scopus (790) Google Scholar, 32.Wang N. Tytell J.D. Ingber D.E. Nat. Rev. Mol. Cell Biol. 2009; 10: 75-82Crossref PubMed Scopus (1239) Google Scholar).A link between structural regulation at the level of signaling cross-talk and the increase in MyD88-dependent activation is consistent with the observed changes in the actin cytoskeleton. Our earlier studies have demonstrated that such Ras-controlled changes in cytoskeletal organization, induced by IL-1, involve phosphorylation of the transmembrane linkage protein talin and alterations at focal adhesions (7.Qwarnström E.E. MacFarlane S.A. Page R.C. Dower S.K. Proc. Natl. Acad. Sci. U.S.A. 1991; 88: 1232-1236Crossref PubMed Scopus (69) Google Scholar, 8.Caunt C.J. Kiss-Toth E. Carlotti F. Chapman R. Qwarnstrom E.E. J. Biol. Chem. 2001; 276: 6280-6288Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). This type of effect on substrate interactions and actin reorganization by cytokines such as TNF has been demonstrated to be adapter-controlled and fundamental in the regulation of biological responses and cell behavior during development and wound healing (33.Cumberbatch M. Dearman R.J. Kimber I. Immunology. 1997; 92: 388-395Crossref PubMed Scopus (292) Google Scholar, 34.Banno T. Gazel A. Blumenberg M. J. Biol. Chem. 2004; 279: 32633-32642Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 35.Haubert D. Gharib N. Rivero F. Wiegmann K. Hösel M. Krönke M. Kashkar H. EMBO J. 2007; 26: 3308-3321Crossref PubMed Scopus (26) Google Scholar). In addition, alterations in cell shape and the actin cytoskeleton will provide an efficient system for controlling NF-κB at the level of the IκBα·NF-κB complex, by allowing rapid changes in the available pool of IκBα during activation (36.Pogson M. Holcombe M. Smallwood R. Qwarnstrom E.E. PLoSOne. 2008; 3: e2367Crossref PubMed Scopus (56) Google Scholar). Future studies will determine the significance of TILRR in the kinetics of complex dissociation, specifically in relation to overall network control and gene induction profiles.We here identify a substrate-dependent and structurally regulated IL-1RI co-receptor, TILRR, which associates with the signaling receptor to potentiate activation of NF-κB. We demonstrate that TILRR association and ensuing changes in receptor complex composition potentiate ligand binding and recruitment of MyD88 and identify a role for TILRR in control of the Ras-GTPase and cytoskeletal organization.We conclude that TILRR controls amplification of inflammatory responses by linking TIR-induced activities with mechanotransduction and expect it to be relevant to a range of diseases characterized by dysregulation of inflammatory responses. Future studies will focus on the role of TILRR in signaling cross-talk and in regulation of gene activation profiles under physiological and pathological conditions. IntroductionImmune and inflammatory response are regulated by Toll-like and IL-1 2The abbreviations used are: ILinterleukinTNFtumor necrosis factorTGFtransforming growth factorMALDI-TOFmatrix-assisted laser desorption/ionization time-of-flightsiRNAsmall interfering RNARTreverse transcriptionEGFPenhanced green fluorescent proteinPBMCperipheral blood mononuclear cell. receptors (1.Akira S. Takeda K. Nat. Rev. Immunol. 2004; 4: 499-511Crossref PubMed Scopus (6586) Google Scholar, 2.Beutler B. Nature. 2004; 430: 257-263Crossref PubMed Scopus (1247) Google Scholar, 3.O'Neill L.A. Curr. Opin. Immunol. 2006; 18: 3-9Crossref PubMed Scopus (516) Google Scholar, 4.Gay N.J. Gangloff M. Annu. Rev. Biochem. 2007; 76: 141-165Crossref PubMed Scopus (505) Google Scholar). Their activation is initiated by ligand binding and co-receptor association to the receptor complex, followed by recruitment of distinct 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.Dinarello C.A. J. Exp. Med. 2005; 201: 1355-1359Crossref PubMed Scopus (402) Google Scholar, 6.Sims J.E. Curr. Opin. Immunol. 2002; 14: 117-122Crossref PubMed Scopus (179) Google Scholar). Its activation is regulated by the proinflammatory cytokines IL-1α and IL-1β and the receptor antagonist, IL-1ra, and by association of the IL-1R accessory protein (AcP) with the receptor complex (6.Sims J.E. Curr. Opin. Immunol. 2002; 14: 117-122Crossref PubMed Scopus (179) Google Scholar). IL-1 responses are, in addition, linked to events related to cell attachment and the cytoskeleton and controlled by the small GTPases (7.Qwarnström E.E. MacFarlane S.A. Page R.C. Dower S.K. Proc. Natl. Acad. Sci. U.S.A. 1991; 88: 1232-1236Crossref PubMed Scopus (69) Google Scholar, 8.Caunt C.J. Kiss-Toth E. Carlotti F. Chapman R. Qwarnstrom E.E. J. Biol. Chem. 2001; 276: 6280-6288Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). 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-κB pathway (1.Akira S. Takeda K. Nat. Rev. Immunol. 2004; 4: 499-511Crossref PubMed Scopus (6586) Google Scholar, 2.Beutler B. Nature. 2004; 430: 257-263Crossref PubMed Scopus (1247) Google Scholar, 3.O'Neill L.A. Curr. Opin. Immunol. 2006; 18: 3-9Crossref PubMed Scopus (516) Google Scholar, 9.Muzio M. Ni J. Feng P. Dixit V.M. Science. 1997; 278: 1612-1615Crossref PubMed Scopus (974) Google Scholar, 10.Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z. Immunity. 1997; 7: 837-847Abstract Full Text Full Text PDF PubMed Scopus (915) Google Scholar).The importance of receptor complex composition and stoichiometry in adapter protein recruitment and TIR signaling is well recognized (4.Gay N.J. Gangloff M. Annu. Rev. Biochem. 2007; 76: 141-165Crossref PubMed Scopus (505) Google Scholar, 11.Gay N.J. Gangloff M. Weber A.N. Nat. Rev. Immunol. 2006; 6: 693-698Crossref PubMed Scopus (154) Google Scholar, 12.Akira S. Yamamoto M. Takeda K. Biochem. Soc. Trans. 2003; 31: 637-642Crossref PubMed Scopus (166) Google Scholar, 13.O'Neill L.A. Bowie A.G. Nat. Rev. Immunol. 2007; 7: 353-364Crossref PubMed Scopus (1971) Google Scholar). 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.Vallés S. Tsoi C. Huang W.Y. Wyllie D. Carlotti F. Askari J.A. Humphries M.J. Dower S.K. Qwarnström E.E. J. Biol. Chem. 1999; 274: 20103-20109Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 15.Valles S. Caunt C.J. Walker M.H. Qwarnstrom E.E. Lab. Invest. 2002; 82: 855-862Crossref PubMed Scopus (3) Google Scholar). 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-κB-regulated inflammatory responses by potentiating recruitment of MyD88 and linking TIR activation with mechanotransduction by controlling induction of the Ras GTPase.
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