Abstract

Lysophosphatidic acid (LPA) is a bioactive lipid composed of a phosphate group, a glycerol backbone, and a single acyl chain that varies in length and saturation. LPA activates six class A G-protein-coupled receptors to provoke various cellular reactions1. Because LPA signalling has been implicated in cancer2 and fibrosis3, the LPA receptors are regarded as promising drug targets. The six LPA receptors are subdivided into the endothelial differentiation gene (EDG) family (LPA1–LPA3)1 and the phylogenetically distant non-EDG family (LPA4–LPA6)4. The structure of LPA1 has enhanced our understanding of the EDG family of LPA receptors5. By contrast, the functional and pharmacological characteristics of the non-EDG family of LPA receptors have remained unknown, owing to the lack of structural information. Although the non-EDG LPA receptors share sequence similarity with the P2Y family of nucleotide receptors4, the LPA recognition mechanism cannot be deduced from the P2Y1 and P2Y12 structures6,7,8 because of the large differences in the chemical structures of their ligands. Here we determine the 3.2 Å crystal structure of LPA6, the gene deletion of which is responsible for congenital hair loss9,10, to clarify the ligand recognition mechanism of the non-EDG family of LPA receptors. Notably, the ligand-binding pocket of LPA6 is laterally open towards the membrane, and the acyl chain of the lipid used for the crystallization is bound within this pocket, indicating the binding mode of the LPA acyl chain. Docking and mutagenesis analyses also indicated that the conserved positively charged residues within the central cavity recognize the phosphate head group of LPA by inducing an inward shift of transmembrane helices 6 and 7, suggesting that the receptor activation is triggered by this conformational rearrangement.

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Acknowledgements

We thank H. Nishimasu for critical comments on the manuscript; T. Nakane for assistance in the diffraction data analyses; K. Ohgomori for technical assistance; A. Inoue for flow cytometry analyses; F. M. N. Kadji for the TGFα shedding assay; Y. Nakamura for the LPA mass spectrometry analysis; and the beamline staff at BL32XU of SPring-8 (Hyogo, Japan) for assistance with data collection. The diffraction experiments were performed at SPring-8 BL32XU (proposals 2015A1024, 2015A1057, 2015B2024, 2015B2057 and 2016A2527), with the approval of RIKEN. This work was supported by grants from the AMED-CREST, by the Platform for Drug Discovery, Informatics and Structural Life Science from AMED, and by a MEXT Grant-in-Aid for Specially Promoted Research (grant 16H06294) to O.N. This work was also supported by JSPS KAKENHI (grant 16J07583 to R.T.; 15H06862 to K.Y.; 17H05000 to T.N.; 16H06574 to R.I.). A.I. was funded by JST, PRESTO (grant JPMJPR1331), and the PRIME from AMED. H.K. and T.N. were funded by JST, PRESTO (grants JPMJPR14L9 and JPMJPR14L8, respectively). J.A. received funding from the AMED-CREST, AMED, and a MEXT Grant-in-Aid for Scientific Research on Innovative Areas (grant 15H05897).

Author information

Author notes

    • Hideaki E. Kato

    Present address: Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA.

Affiliations

  1. Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan

    • Reiya Taniguchi
    • , Hideaki E. Kato
    • , Tomohiro Nishizawa
    • , Ryuichiro Ishitani
    •  & Osamu Nureki
  2. Global Research Cluster, RIKEN, Wako, Saitama 351-0198, Japan

    • Reiya Taniguchi
    • , Hideaki E. Kato
    • , Tomohiro Nishizawa
    • , Ryuichiro Ishitani
    •  & Osamu Nureki
  3. Laboratory of Molecular and Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan

    • Asuka Inoue
    • , Akiharu Uwamizu
    •  & Junken Aoki
  4. Japan Science and Technology Agency (JST), Precursory Research for Embryonic Science and Technology (PRESTO), Kawaguchi, Saitama 332-0012, Japan

    • Asuka Inoue
    • , Kunio Hirata
    •  & Tomohiro Nishizawa
  5. Laboratory of Organic and Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan

    • Misa Sayama
    • , Yuko Otani
    •  & Tomohiko Ohwada
  6. RIKEN SPring-8 Center, Sayo-gun, Hyogo 679-5148, Japan

    • Keitaro Yamashita
    •  & Kunio Hirata
  7. Laboratory of Heterocyclic Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan

    • Masahito Yoshida
    •  & Takayuki Doi
  8. Department of Systems Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan

    • Yoshiki Tanaka
  9. Department of Cell Biology, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan

    • Yoshiko Nakada-Nakura
  10. Japan Agency for Medical Research and Development, Core Research for Evolutional Science and Technology (AMED-CREST), Chiyoda-ku, Tokyo 100-0004, Japan

    • Junken Aoki

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Contributions

R.T. purified and crystallized LPA6, determined the structure, planned the mutational analyses, and performed the ligand-binding assays and thermostability measurements. A.I. and A.U. performed the TGFα shedding assays, flow cytometry experiments, and analysed the data. M.S. performed the ligand-docking simulations under the supervision of Y.O. and T.O. K.Y. and K.H. assisted with the diffraction data collection and the data analyses. M.Y. performed the ligand synthesis under the supervision of T.D. Y.T. performed the preliminary expression check of LPA6. H.E.K. and Y.N.-N. assisted with the structure determination. T.N. assisted with the data analyses. R.T., A.I., M.S., T.N., J.A. and O.N. wrote the manuscript, with feedback from all of the authors. T.O., R.I., J.A. and O.N. supervised the research.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Junken Aoki or Osamu Nureki.

Reviewer Information Nature thanks T. Shimizu, I. von Kügelgen and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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