Isoform- and ligand-specific modulation of the adhesion GPCR ADGRL3/Latrophilin3 by a synthetic binder

Creators: Kordon, Szymon P.; Dutka, Przemysław; Adamska, Justyna M.; Bandekar, Sumit J.; Leon, Katherine; Erramilli, Satchal K.; Adams, Brock; Li, Jingxian; Kossiakoff, Anthony A.; Araç, Demet

Abstract

Adhesion G protein-coupled receptors (aGPCRs) are cell-surface proteins with large extracellular regions that bind to multiple ligands to regulate key biological functions including neurodevelopment and organogenesis. Modulating a single function of a specific aGPCR isoform while affecting no other function and no other receptor is not trivial. Here, we engineered an antibody, termed LK30, that binds to the extracellular region of the aGPCR ADGRL3, and specifically acts as an agonist for ADGRL3 but not for its isoform, ADGRL1. The LK30/ADGRL3 complex structure revealed that the LK30 binding site on ADGRL3 overlaps with the binding site for an ADGRL3 ligand – teneurin. In cellular-adhesion assays, LK30 specifically broke the trans-cellular interaction of ADGRL3 with teneurin, but not with another ADGRL3 ligand – FLRT3. Our work provides proof of concept for the modulation of isoform- and ligand-specific aGPCR functions using unique tools, and thus establishes a foundation for the development of fine-tuned aGPCR-targeted therapeutics.

Additional Information

This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. We thank Engin Ӧzkan for the use of flow cytometer and all members of the Araç lab for helpful discussions. We also thank the staff at the Advanced Photon Source (APS) at Argonne National Labs (ANL). GM/CA@APS has been funded by the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006, P30GM138396). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. The Eiger 16 M detector at GM/CA-XSD was funded by NIH grant S10 OD012289. This work was supported by grants R35 GM148412 (to D.A.), R01 GM134035-01 (to D.A.), F32 GM142266 (to S.J.B.) and R01 GM117372 (to A.A.K.). Contributions. S.P.K. and D.A. designed all experiments and interpreted results. S.P.K. expressed and purified all proteins (with assistance from B.A. and K.L.). P.D. carried out phage display selection and sABs characterization. S.K.E. and S.P.K. assisted with SPR experiments. S.P.K. and J.M.A. performed flow cytometry and cell-based signaling assays. S.J.B. and S.P.K. performed crystallography experiments (with assistance from B.A.) and structure determination. J.L. participated in cell-cell aggregation experiments. S.P.K. and D.A. wrote the manuscript with assistance from A.A.K., S.J.B. and P.D.. D.A. and A.A.K. supervised the project. Data availability The coordinates for the crystal structure of ADGRL3/LK30 generated in this study have been deposited in the Protein Data Bank [http://www.rcsb.org] under accession code PDB 8DJG. All the other relevant structures referenced in this work are available under the accession codes 6VHH, 4XWO, 5AFB, 5UCB, 6SKA and 5FTU. The authors declare that all data supporting the findings of this study are available within the article and the source data underlying Figs. 1b, g, 2a-c, 5e, j and Supplementary Figs. 4, 5 and 8 are provided as a Source Data file. The gating strategy for flow cytometry experiments can be found in Supplementary Fig. 10. Source data are provided with this paper. The authors declare no competing interests.

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