TIRAP, an Adaptor Protein for TLR2/4, Transduces a Signal from RAGE Phosphorylated upon Ligand Binding Masakiyo Sakaguchi1, Hitoshi Murata1, Ken-ichi Yamamoto1, Tomoyuki Ono 1 , Yoshihiko Sakaguchi2, Akira Motoyama3, Toshihiko Hibino 3 , Ken Kataoka1, Nam-ho Huh 1 * 1 Department of Cell Biology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Kita-ku, Okayama, Japan, 2 Interdisciplinary Research Organization, University of Miyazaki, Kiyotakecho, Miyazaki, Japan, 3 Shiseido Research Center, Kanazawa-ku, Yokohama, Japan Abstract The receptor for advanced glycation end products (RAGE) is thought to be involved in the pathogenesis of a broad range of inflammatory, degenerative and hyperproliferative diseases. It binds to diverse ligands and activates multiple intracellular signaling pathways. Despite these pivotal functions, molecular events just downstream of ligand-activated RAGE have been surprisingly unknown. Here we show that the cytoplasmic domain of RAGE is phosphorylated at Ser391 by PKCf upon binding of ligands. TIRAP and MyD88, which are known to be adaptor proteins for Toll-like receptor-2 and -4 (TLR2/4), bound to the phosphorylated RAGE and transduced a signal to downstream molecules. Blocking of the function of TIRAP and MyD88 largely abrogated intracellular signaling from ligand-activated RAGE. Our findings indicate that functional interaction between RAGE and TLRs coordinately regulates inflammation, immune response and other cellular functions. Citation: Sakaguchi M, Murata H, Yamamoto K-i, Ono T, Sakaguchi Y, et al. (2011) TIRAP, an Adaptor Protein for TLR2/4, Transduces a Signal from RAGE Phosphorylated upon Ligand Binding. PLoS ONE 6(8): e23132. doi:10.1371/journal.pone.0023132 Editor: Dhyan Chandra, Roswell Park Cancer Institute, United States of America Received March 18, 2011; Accepted July 8, 2011; Published August 1, 2011 Copyright: 2011 Sakaguchi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by grants from the Ministry of Health, Labor and Welfare (Research for Intractable Diseases) (N. Huh), from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Grant–in-Aid for Young Scientists (A)) (M. Sakaguchi), and from Takeda Science Foundation (M. Sakaguchi). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study. Akira Motoyama and Toshihiko Hibino are employees of the Shiseido Research Center who played a role in study design, data collection and analysis, decision to publish and preparation of the manuscript. Competing Interests: Akira Motoyama and Toshihiko Hibino are employees of the Shiseido Research Center. There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: namu@md.okayama-u.ac.jp Introduction The receptor for advanced glycation end products (RAGE) is a type I single-pass transmembrane protein belonging to the immunoglobulin superfamily [1]. RAGE is thought to be involved in a broad range of inflammatory, degenerative and hyperproli- ferative diseases, including sepsis, rheumatoid arthritis, diabetic nephropathy, atherosclerosis, cancer, and neurological disorders [2–5]. The receptor is composed of an extracellular region (one V- type and two C-type immunoglobulin domains), a hydrophobic transmembrane-spanning domain, and a short cytoplasmic tail. The extracellular domain can bind diverse ligands, including advanced glycation end products (AGEs) [2], high-mobility group box 1 (HMGB1) [6], S100 family proteins [7], and amyloid fibrils [8]. Binding of ligands to RAGE leads to activation of multiple signaling pathways, including signaling pathways involved in the production of reactive oxygen species, Ras-mediated extracellular signal-regulated kinase 1/2 (ERK1/2), Akt, stress-activated protein kinase/c-Jun-NH2-terminal kinase (SAPK/JNK), p38 MAP kinase, small GTPase Cdc42/Rac1, and NFkB and AP-1 transcription factors [9–13]. In accordance with this, cellular biological outcomes are also diverse and include induction of apoptosis, hyperproliferation, increased motility, and production of inflammatory cytokines [4,5,14]. It is intriguing that different RAGE ligands often show different effects in a given type of cell. For example, we previously showed that dimerized S100A11 activated Akt and induced production of EGF, thus stimulating growth of normal human keratinocytes via RAGE [15]. In the same cells, S100A8/A9, which is known to be a RAGE ligand [16], enhanced production of proinflammatory cytokines includ- ing IL-8, IL-1F9 and TNFa but neither induced EGF nor activated Akt [17]. RAGE has a short cytoplasmic domain (41 amino acids), from which RAGE-triggered signaling is initiated. The cytoplasmic domain has no obvious signaling domain or any motifs known for receptor signaling. Hudson et al. identified Diaphanous-1 (Dia-1) to be a binding partner to the RAGE cytoplasmic domain by employing a yeast two-hybrid method [18]. Dia-1 is required for controlling the activation of Rac-1 and Cdc42, resulting in promotion of cellular migration. It is unclear, however, whether diverse downstream signaling pathways of RAGE are controlled by Dia-1 alone. Ishihara et al. [19] reported that extracellular signal-regulated protein kinase-1 and -2 (ERK1/2) bind to the cytoplamic domain of RAGE, but the biological significance of the interaction was not clarified. It remains a major challenge to reveal how the cytoplasmic domain of RAGE is activated upon ligand binding and how its activation is transduced to downstream signaling molecules for biological significance of RAGE in normal and pathological circumstances. In the present study, we focused on molecular events in and at the cytoplasmic domain of RAGE. We found that the cytoplasmic domain of RAGE was phosphorylated at Ser391 by PKCf upon binding of ligands. To our surprise, TIRAP and MyD88, which PLoS ONE | www.plosone.org 1 August 2011 | Volume 6 | Issue 8 | e23132