Abstract
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.
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Acknowledgements
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.
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Contributions
J.M. developed the FRET-based detectors and the FRET-based conformational change sensors and performed sensor experiments, IP1 assays, co-immunoprecipitation and immunoblotting. C.X. proposed the idea and set up the protocol for the nanobody-based sensors for the detection of mGlu2 expression and activation in dissociated brain cells and performed the DN1 and DN10 sensor experiments in HEK293 cells and dissociated brain cells and IP1 assays. P.-A.L. performed tissue immunofluorescence assays. S.R. designed and performed experiments with the FRET-based detectors and conformational change sensors. M.M. performed TR-FRET microscopy imaging, nanobody production, purification and labeling. R.Z. performed nanobody production, purification, labeling and co-immunoprecipitation. P.S. and E.B. developed the FRET-based conformational change biosensor for the mGlu2 homodimer in HEK293 cells. J.A.J.B. and J.L.M. provided brain samples and trained J.M. for brain sample preparation. J.G.-M. prepared brain samples for wild-type and mGlu2-KO mice. P.C. screened for anti-mGlu2 and anti-mGlu4 nanobodies and prepared the Fc versions. J.L., J.-P.P. and P.R. conceived experiments, supervised the work and wrote the manuscript.
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P.R. and J.-P.P. are funded by PerkinElmer Cisbio through the collaborative laboratory Eidos. The remaining authors declare no competing interests.
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Extended data
Extended Data Fig. 1 DN1/DN10 FRET-based biosensor detects mGlu2 homodimers on transfected HEK-293 cells.
(a) TR-FRET signal measured on HEK-293 cells transiently transfected with mGlu2 receptors in the presence of donor nanobody DN1-Tb (7.5 nM) and LY379268 (1 μM), with or without acceptor nanobody DN10-d2 (15 nM). (b) Specific TR-FRET signal on HEK-293 cells transiently transfected with mGlu2 receptors in the presence of DN1-Tb (7.5 nM) with increasing concentrations of DN10-d2. For each concentration of acceptor nanobody, specific FRET signal was the difference between samples in the presence of LY379268 (1 μM) and LY341495 (10 μM). Data are mean ± SEM of three independent experiments performed in triplicates and normalized to the maximum. (c) TR-FRET signal measured on HEK-293 cells transiently transfected with indicated mGlu receptors or on mock cells with LY379268 (1 μM). In a and c, data are mean ± SEM of triplicate determinations from one representative out of three experiments.
Extended Data Fig. 2 TR-FRET confocal imaging on primary cultured hippocampal neurons.
DN10-Tb (80 nM) and DN1-d2 (120 nM) were applied on the primary cultured hippocampal neurons. Fluorescence signals of different channels, TR-FRET channel (left), donor channel (middle) or acceptor channel (right) were obtained under agonist (150 nM LY379268, top) or antagonist (1 μM LY341495, bottom). Scale bar: 20 μm.
Extended Data Fig. 3 DN1/DN10 biosensor measures endogenous mGlu2 on cells from brain regions.
(a) Diagram shows flow of the tissue samples experiments. (b) Schematic diagram of adult mouse brain removed from most of the cortex, in top view. Brain regions analyzed are olfactory bulb (blue), prefrontal cortex (red), striatum (green), hippocampus (purple), midbrain (orange) and cerebellum (gray). (c) Relative quantification of the endogenous mGlu2 receptor in the indicated brain regions in the same mouse using the indicate FRET-based biosensor in presence of 1 μM LY379268. Data are mean ± SEM of triplicate determinations from one representative out of three experiments.
Extended Data Fig. 4 DN1-d2 immunofluorescence staining of a saggital section in wild-type and mGlu2 knock out mouse brains.
The scale bars are indicated.
Extended Data Fig. 5 FRET-based detector mainly detects mGlu2 homodimer.
(a) TR-FRET signal measured on HEK-293 cells transiently transfected with mGlu2 receptors in the presence of DN1-Tb (25 nM) and LY341495 (10 μM), with or without DN1-d2 (25 nM). (b) Specific TR-FRET signal on HEK-293 cells transiently transfected with mGlu2 receptors in the presence of DN1-Tb (25 nM) and LY341495 (10 μM) with increasing concentrations of DN1-d2. For each concentration of acceptor nanobody, specific FRET signal was the difference between samples transfected with mGlu2 receptor or not. Data are mean ± SEM of three independent experiments performed in triplicates and normalized to the maximum. (c and d) TR-FRET intensity and cell surface expression were measured on various expression levels of the indicated cell samples. Experiments were performed with HEK-293 cells transiently transfected with SNAPmGlu2 which fused a SNAP tag at the N terminus of receptors (c) or transiently co-transfected with SNAPmGlu2C1 and mGlu2C2 or mGlu4C2 (d). The surface expression of receptors was measured as the specific Tb emission at 620 nm after labeling by substrate SNAP-Lumi4-Tb and exciting at 337 nm. TR-FRET intensity was measured in the presence of 10 μM LY341495. (e) Relative quantification of mGlu2 receptor in the indicated brain tissues from a same mouse in the presence of 10 μM LY341495. For a and c-e, Data are mean ± SEM of triplicate determinations from one representative out of three experiments.
Extended Data Fig. 6 Nanobody DN42 specifically targets mGlu4 receptors.
(a) Cartoon illustrating the principle of the TR-FRET binding assay. The receptor fused to a SNAP-tag (dark gray circled labeled ‘S’) is labeled with donor fluorescent dye Lumi4-Tb (blue circled ‘D’) while the nanobody DN42 (dark blue) bearing a 6xHis tag epitope at its C-terminus is labeled with 100 nM of anti-His antibody (bright blue) coupled to d2 fluorophores (red circled ‘A’). Binding of the nanobody to the receptor is then measured by TR-FRET. (b) Specific TR-FRET binding data obtained with the indicated mGlu receptor and either 100 nM Fc-DN42 or a control irrelevant nanobody (100 nM) in cells under indicated drug conditions, in the presence of the agonist 1 μM quisqualic acid (mGlu1 and 5) or 100 μM L-AP4 (mGlu4, 6, 7 and 8) or 100 nM LY379268 (mGlu2 and 3) or the antagonist LY341495 (10 μM). Data are mean ± SEM of triplicate determinations from one representative out of three experiments. (c) Saturation binding curves obtained with DN42 on SNAPmGlu4 receptors under indicated drug conditions, in the presence of the agonist L-AP4 (100 μM), or the antagonist LY341495 (10 μM). Data are mean ± SEM of three independent experiments performed in triplicates and normalized to the maximum. (d) DN42-d2 immunofluorescence staining of a sagittal section in wild-type and mGlu4 KO mouse brains. The scale bars are indicated.
Extended Data Fig. 7 FRET-based detector mainly detects mGlu4 homodimer.
(a) TR-FRET signal measured on HEK-293 cells transiently transfected with mGlu4 receptors in the presence of DN42-Tb (1.6 nM) and LY341495 (10 μM), with or without DN42-d2 (1.6 nM). (b) Specific TR-FRET signal on HEK-293 cells transiently transfected with mGlu4 receptors in the presence of DN42-Tb (1.6 nM) and LY341495 (10 μM), with increasing concentrations of DN42-d2. For each concentration of acceptor nanobody, specific FRET signal was the difference between samples transfected with mGlu4 receptors or not. Data are mean ± SEM of three independent experiments performed in triplicates and normalized to the maximum. (c and d) TR-FRET intensity and cell surface expression were measured on various expression levels of the indicated cell samples. Experiments were performed with HEK-293 cells transiently transfected with SNAPmGlu4 which fused a SNAP tag at the N terminus of receptors (c) or transiently co-transfected with SNAPmGlu4C2 and mGlu4C1 or mGlu2C1 (d). The surface expression of receptors was measured as the specific Tb emission at 620 nm after labeling by substrate SNAP-Lumi4-Tb and exciting at 337 nm. TR-FRET intensity was measured in the presence of 10 μM LY341495. (e) Relative quantification of mGlu4 receptor in the indicated brain tissues from a same mouse in the presence of 10 μM LY341495. For a and c-e, Data are mean ± SEM of triplicate determinations from one representative out of three experiments.
Extended Data Fig. 8 FRET-based detector only detects mGlu2-4 heteromer.
(a) TR-FRET signal measured on HEK-293 cells transiently expressing mGlu2C1-4C2 heterodimer in the presence of DN42-Tb (1.6 nM) and LY341495 (10 μM), with or without DN1-d2 (25 nM). (b) Specific TR-FRET signal on HEK-293 cells transiently expressing mGlu2C1-4C2 heterodimer in the presence of DN42-Tb (1.6 nM) and LY341495 (10 μM) with increasing concentrations of DN1-d2. For each concentration of acceptor nanobody, specific FRET signal was the difference between samples expressing mGlu2C1-4C2 heterodimer or not. Data are mean ± SEM of three independent experiments performed in triplicates and normalized to the maximum. (c) TR-FRET intensity and cell surface expression were measured for various expression levels of the indicated cell samples. Experiments were performed with HEK-293 cells transiently co-transfected with SNAPmGlu2C1 and mGlu4C2 (red) or SNAPmGlu2C1 and mGlu2C2 (blue). The surface expression of receptors was measured as the specific Tb emission at 620 nm after labeling by substrate SNAP-Lumi4-Tb and exciting at 337 nm. TR-FRET intensity was measured in the presence of 10 μM LY341495. (d) TR-FRET signal measured on HEK-293 cells transiently transfected with indicated the mGlu receptors or on mock cells with LY341495 (10 μM). (e and g) Relative quantification of mGlu2-4 heterodimer in the indicated brain tissues in the presence of 10 μM LY341495. (f) mGlu2 and mGlu4 are co-immunoprecipitated from mouse olfactory bulb. FcDN42 was used as probe antibody in pull down experiments. FcGFP was used as a negative control (IgG control). Results are one representative out of three independent experiments performed. For a, c-e and g, Data are mean ± SEM of triplicate determinations from one representative out of three experiments.
Extended Data Fig. 9 Nanobody DN10 allows to develop a FRET-based conformational biosensor for mGlu2-4 heterodimer.
(a) Schematic representation of the TR-FRET binding assay for the mGlu2-4 controlled heterodimer. The receptor fused to a SNAP-tag (dark gray circled labeled ‘S’) at the N-terminus of mGlu4 subunit is labeled with donor fluorescent dye Lumi4-Tb (blue circled ‘D’), while the nanobody DN10 (purple) coupled to a d2 fluorophore (red circled ‘A’). Binding of the nanobody to the receptor is then measured by TR-FRET. (b) Saturation binding experiments with DN10-d2 on the cell surface mGlu2-4 controlled heterodimer using the constructs mGlu2C1 and SNAPmGlu4C2 transiently co-transfected in HEK-293 cells. Binding of DN10-d2 to the receptor was measured by TR-FRET under the indicated conditions, buffer (gray, n = 5) or in the presence of the mGlu2 agonist LY379268 (1 μM, blue, n = 5), the mGlu4 agonist L-AP4 (100 μM, red, n = 3), or the antagonist LY341495 (10 μM, black, n = 5). Data are mean ± SEM of at least three individual experiments each performed in triplicates and normalized to the maximum. (c and d) Dose-dependent effects of the indicated ligands on TR-FRET signal between SNAP-tag labeled with donor fluorescent dye Lumi4-Tb (blue circled ‘D’) and 25 nM DN10-d2. Signals were measured on HEK-293 cells transiently co-transfected with mGlu2C1 and SNAPmGlu4C2 (c, n = 3) or co-transfected with mGlu2C1 and SNAPmGlu2C2 (d, n = 4). Data are mean ± SEM of at least three independent experiments performed in triplicate and normalized to the response of LY379268. (e) The FRET potencies (pEC50) in the indicated conditions. Data are mean ± SEM from HEK-293 cells transiently co-transfected with mGlu2C1 and SNAPmGlu4C2 (c, n = 3) or with mGlu2C1 and SNAPmGlu2C2 (d, n = 4). One-way ANOVA with Tukey’s multiple comparisons test (mGlu2 under the LY379268 is not included), with **P = 0.0041. (f) TR-FRET signal measured on HEK-293 cells transiently transfected with mGlu2C1 and mGlu4C2 or on mock cells in presence of DN42-Tb (1.6 nM) and DN10-d2 (25 nM) with 1 μM LY379268 (LY37) or 10 μM LY341495 (LY34). (g) TR-FRET signal measured on HEK-293 cells transiently transfected with the indicated mGlu receptors or on mock cells with LY379268 (1 μM). For f - g, Data are mean ± SEM of triplicate determinations from one representative out of three experiments.
Extended Data Fig. 10 IP1 accumulation of mGlu2-4 heterodimer shows its specific pharmacological fingerprints.
(a and b) Dose-dependent effects of the indicated ligands on IP1 accumulation. Signals were measured on HEK-293 cells transiently co-transfected with mGlu2C1 and mGlu4C2 (a) or with mGlu2 and mGlu4 (b) and EAAC1 and the chimeric G protein Gqi9. Data are mean ± SEM of three independent experiments performed in triplicate and normalized to the response of LY379268. (c) IP1 accumulation potencies (pEC50) of the indicated drug on the receptor conditions in a and b. Data are mean ± SEM of three independent experiments. Two-way ANOVA with Tukey’s multiple comparisons test, with ***P < 0.001 and *P ≤ 0.05. (d) TR-FRET potencies (pEC50) for the indicated ligands on the conformational sensor with the constructs mGlu2C1 and mGlu4C2, or when both wild-type mGlu2 and mGlu4 subunits are co-transfected. Data are presented as the mean ± SEM of four independent experiments. Two-way ANOVA followed by a Tukey’s post-hoc test, with ***P < 0.001, **P ≤ 0.01, and ns P > 0.05. (e) TR-FRET potencies (pEC50) of the ligands on the conformational sensor measured in dissociated olfactory bulb cells. Data are presented as the mean ± SEM of four independent experiments. One-way ANOVA followed by a Tukey’s post-hoc test, with ****P ≤ 0.0001 and ***P ≤ 0.001. (f) Correlation between the potencies (pEC50) determined with the indicated agonist conditions by the heterodimer conformational sensor and IP1 assay. Data are mean ± SEM of at least three independent experiments (TR-FRET of olfactory bulb (n = 4), mGlu2 + mGlu4 (n = 3) and mGlu2C1 + mGlu4C2 (n = 3) and IP1 accumulation of mGlu2C1 + mGlu4C2 (n = 3)). LY37 means LY379268. All exact P values are indicated in the panels c-e.
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Meng, J., Xu, C., Lafon, PA. et al. Nanobody-based sensors reveal a high proportion of mGlu heterodimers in the brain. Nat Chem Biol 18, 894–903 (2022). https://doi.org/10.1038/s41589-022-01050-2
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DOI: https://doi.org/10.1038/s41589-022-01050-2
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