TY - JOUR
T1 - Nanobody-based sensors reveal a high proportion of mGlu heterodimers in the brain
AU - Meng, Jiyong
AU - Xu, Chanjuan
AU - Lafon, Pierre André
AU - Roux, Salomé
AU - Mathieu, Michaël
AU - Zhou, Rui
AU - Scholler, Pauline
AU - Blanc, Emilie
AU - Becker, Jérôme A.J.
AU - Le Merrer, Julie
AU - González-Maeso, Javier
AU - Chames, Patrick
AU - Liu, Jianfeng
AU - Pin, Jean Philippe
AU - Rondard, Philippe
N1 - Funding Information:
We thank D. Nevoltris and D. Meyer (IRCM) for screening nanobodies and preparing the Fc-DN42 construct, respectively. We thank the Arpège platform of the Institut de Génomique Fonctionnelle for providing facilities and technical support, the imaging facility Montpellier Ressources Imagerie (MRI), member of the national infrastructure France-BioImaging supported by the French National Research Agency (ANR-10-INBS-04, Investments for the Future), the animal facility RAM-iExplore from BioCampus and PerkinElmer Cisbio for providing reagents. We thank C. Goudet and T. Durroux (IGF) for providing the mGlu4-KO mice and for assistance with TR-FRET microscopy, respectively. J.M. and C.X. were supported by the Sino-French Cai Yuanpei program (grant nos. 201604490217 and 201304490188, respectively). P.R. and J.-P.P. were supported by the Centre National de la Recherche Scientifique (CNRS, PICS no. 07030, PRC no. 1403), the Institut National de la Santé et de la Recherche Médicale (INSERM, IRP BrainSignal), the Fondation pour la Recherche Médicale (equipe DEQ20170336747), the Eidos collaborative laboratory with PerkinElmer Cisbio, the Franco–Chinese Joint Scientific and Technological Commission (CoMix) from the French Embassy in China and the LabEx MAbImprove (grant NR-10-LABX-5301). P.R. and J.-P.P. were supported by the ANR (grants ANR-15-CE18-0020-01, ANR-20-CE18-0011-01 and ANR-20-CE44-0006-02). J.L. was supported by the Program of Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory, grant no. 2010A080813001), the Ministry of Science and Technology of China (grant no. 2018YFA0507003), the National Natural Science Foundation of China (grant nos. 81720108031, 31721002 and 81872945), the Program for Introducing Talents of Discipline to the Universities of the Ministry of Education (grant no. B08029), the Key Program of Natural Science Foundation of Hubei Province (grant no. 2019ACA128) and Wuhan (2019020701011481). P.C. was supported by the FUI of the French government (FUI, Cell2Lead project). J.G.-M. was supported by National Institutes of Health grants R01MH084894 and R01MH111940. The mouse pictures in Fig. 1 and Extended Data Fig. 3a were modified from Servier Medical Art (http://smart.servier.com/ ), licensed under a Creative Commons Attribution 3.0 generic license (https://creativecommons.org/licenses/by/3.0/ ). The mouse brain in Extended Data Fig. 3a is from Figdraw (https://www.figdraw.com/ ). Pictures of the microplate in Fig. 1 and Extended Data Fig. 3a are from PerkinElmer Cisbio.
Funding Information:
We thank D. Nevoltris and D. Meyer (IRCM) for screening nanobodies and preparing the Fc-DN42 construct, respectively. We thank the Arpège platform of the Institut de Génomique Fonctionnelle for providing facilities and technical support, the imaging facility Montpellier Ressources Imagerie (MRI), member of the national infrastructure France-BioImaging supported by the French National Research Agency (ANR-10-INBS-04, Investments for the Future), the animal facility RAM-iExplore from BioCampus and PerkinElmer Cisbio for providing reagents. We thank C. Goudet and T. Durroux (IGF) for providing the mGlu4-KO mice and for assistance with TR-FRET microscopy, respectively. J.M. and C.X. were supported by the Sino-French Cai Yuanpei program (grant nos. 201604490217 and 201304490188, respectively). P.R. and J.-P.P. were supported by the Centre National de la Recherche Scientifique (CNRS, PICS no. 07030, PRC no. 1403), the Institut National de la Santé et de la Recherche Médicale (INSERM, IRP BrainSignal), the Fondation pour la Recherche Médicale (equipe DEQ20170336747), the Eidos collaborative laboratory with PerkinElmer Cisbio, the Franco–Chinese Joint Scientific and Technological Commission (CoMix) from the French Embassy in China and the LabEx MAbImprove (grant NR-10-LABX-5301). P.R. and J.-P.P. were supported by the ANR (grants ANR-15-CE18-0020-01, ANR-20-CE18-0011-01 and ANR-20-CE44-0006-02). J.L. was supported by the Program of Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory, grant no. 2010A080813001), the Ministry of Science and Technology of China (grant no. 2018YFA0507003), the National Natural Science Foundation of China (grant nos. 81720108031, 31721002 and 81872945), the Program for Introducing Talents of Discipline to the Universities of the Ministry of Education (grant no. B08029), the Key Program of Natural Science Foundation of Hubei Province (grant no. 2019ACA128) and Wuhan (2019020701011481). P.C. was supported by the FUI of the French government (FUI, Cell2Lead project). J.G.-M. was supported by National Institutes of Health grants R01MH084894 and R01MH111940. The mouse pictures in Fig. and Extended Data Fig. were modified from Servier Medical Art ( http://smart.servier.com/ ), licensed under a Creative Commons Attribution 3.0 generic license ( https://creativecommons.org/licenses/by/3.0/ ). The mouse brain in Extended Data Fig. is from Figdraw ( https://www.figdraw.com/ ). Pictures of the microplate in Fig. and Extended Data Fig. are from PerkinElmer Cisbio.
Publisher Copyright:
© 2022, The Author(s), under exclusive licence to Springer Nature America, Inc.
PY - 2022/8
Y1 - 2022/8
N2 - Membrane proteins, including ion channels, receptors and transporters, are often composed of multiple subunits and can form large complexes. Their specific composition in native tissues is difficult to determine and remains largely unknown. In this study, we developed a method for determining the subunit composition of endogenous cell surface protein complexes from isolated native tissues. Our method relies on nanobody-based sensors, which enable proximity detection between subunits in time-resolved Förster resonance energy transfer (FRET) measurements. Additionally, given conformation-specific nanobodies, the activation of these complexes can be recorded in native brain tissue. Applied to the metabotropic glutamate receptors in different brain regions, this approach revealed the clear existence of functional metabotropic glutamate (mGlu)2–mGlu4 heterodimers in addition to mGlu2 and mGlu4 homodimers. Strikingly, the mGlu4 subunits appear to be mainly heterodimers in the brain. Overall, these versatile biosensors can determine the presence and activity of endogenous membrane proteins in native tissues with high fidelity and convenience. [Figure not available: see fulltext.]
AB - Membrane proteins, including ion channels, receptors and transporters, are often composed of multiple subunits and can form large complexes. Their specific composition in native tissues is difficult to determine and remains largely unknown. In this study, we developed a method for determining the subunit composition of endogenous cell surface protein complexes from isolated native tissues. Our method relies on nanobody-based sensors, which enable proximity detection between subunits in time-resolved Förster resonance energy transfer (FRET) measurements. Additionally, given conformation-specific nanobodies, the activation of these complexes can be recorded in native brain tissue. Applied to the metabotropic glutamate receptors in different brain regions, this approach revealed the clear existence of functional metabotropic glutamate (mGlu)2–mGlu4 heterodimers in addition to mGlu2 and mGlu4 homodimers. Strikingly, the mGlu4 subunits appear to be mainly heterodimers in the brain. Overall, these versatile biosensors can determine the presence and activity of endogenous membrane proteins in native tissues with high fidelity and convenience. [Figure not available: see fulltext.]
UR - http://www.scopus.com/inward/record.url?scp=85131567466&partnerID=8YFLogxK
U2 - 10.1038/s41589-022-01050-2
DO - 10.1038/s41589-022-01050-2
M3 - Article
AN - SCOPUS:85131567466
VL - 18
SP - 894
EP - 903
JO - Nature Chemical Biology
JF - Nature Chemical Biology
SN - 1552-4450
IS - 8
ER -