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Published August 21, 2018 | Supplemental Material
Journal Article Open

Stepwise phosphorylation of leukotriene B_4 receptor 1 defines cellular responses to leukotriene B_4

Abstract

Leukotriene B_4 (LTB_4) receptor type 1 (BLT1) is abundant in phagocytic and immune cells and plays crucial roles in various inflammatory diseases. BLT1 is phosphorylated at several serine and threonine residues upon stimulation with the inflammatory lipid LTB_4. Using Phos-tag gel electrophoresis to separate differentially phosphorylated forms of BLT1, we identified two distinct types of phosphorylation, basal and ligand-induced, in the carboxyl terminus of human BLT1. In the absence of LTB_4, the basal phosphorylation sites were modified to various degrees, giving rise to many different phosphorylated forms of BLT1. Different concentrations of LTB_4 induced distinct phosphorylation events, and these ligand-induced modifications facilitated additional phosphorylation events at the basal phosphorylation sites. Because neutrophils migrate toward inflammatory sites along a gradient of LTB_4, the degree of BLT1 phosphorylation likely increases in parallel with the increase in LTB_4 concentration as the cells migrate. At high concentrations of LTB_4, deficiencies in these two types of phosphorylation events impaired chemotaxis and β-hexosaminidase release, a proxy for degranulation, in Chinese hamster ovary (CHO-K1) and rat basophilic leukemia (RBL-2H3) cells, respectively. These results suggest that an LTB_4 gradient around inflammatory sites enhances BLT1 phosphorylation in a stepwise manner to facilitate the precise migration of phagocytic and immune cells and the initiation of local responses, including degranulation.

Additional Information

© 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Submitted 1 August 2017; Accepted 26 July 2018; Published 21 August 2018. We thank S. Higashiyama (Ehime University Proteo-Science Center) for providing the pRc-CMV/AP-TGFα plasmid to construct the TGFα shedding assay system and J.-i. Miyazaki (Osaka University) for providing the pCAGGS vector to develop the NanoBiT–β-arrestin recruitment assay. We also thank A. Kikuchi, M. Segawa (Okayama University of Science), and Y. Aruga (The University of Tokyo) for their research project works. We are grateful to all laboratory members for their constructive comments. Funding: This work was supported by MEXT (Ministry of Education, Culture, Sports, Science and Technology)/Japan Society for the Promotion of Science KAKENHI grant numbers 26460061 (to M.N.), 15H05897 (to J.A.), 15H02319 (to T.N.), 15H05901, 15H05904, and 15H04708 (to T.Y.) and by the Ryobi Teien Memory Foundation (to M.N.), the Naito Foundation (to M.N.), the Mitsubishi Foundation (to M.N.), the Takeda Science Foundation (to M.N. and T.S.), the Science Research Promotion fund by The Promotion and Mutual Aid Corporation for Private Schools of Japan (to M.N.), JST (Japan Science and Technology Agency) grant number JPMJPR1331 (to A.I.), and AMED (Japan Agency for Medical Research and Development) grant numbers JP17gm5910013 (to A.I.) and JP17gm0710001 (to J.A.). Department of Lipid Signaling at the National Center for Global Health and Medicine is financially supported by ONO Pharmaceutical Co. Ltd. (T.S.). Author contributions: Y.N., K.Y., I.T., J.-i.Y., and N.M. performed the experiments. M.T., S. Yamahira, S. Yamaguchi, and T.N. performed SrtA-mediated labeling of BLT1 and analyzed the trafficking of the receptor. T.I. and T.Y. performed a chemotaxis assay using TAXIScan-FL. A.I. and J.A. carried out the β-arrestin–binding assay using the NanoBiT system. Y.N., M.T., T.I., A.I., and M.N. performed statistical analyses. T.S. and M.N. conceived and designed the study and acquired, analyzed, and interpreted the data. Y.N. and M.N. prepared the manuscript. Competing interests: The authors declare that they have no competing financial interests. Data and materials availability: All data needed to evaluate the conclusions in this paper are present in the paper or in the Supplementary Materials. Plasmids require a material transfer agreement from Okayama University of Science.

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August 19, 2023
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October 18, 2023