Single-molecule FRET imaging of GPCR dimers in living cells.

Dimerization Fluorescence Resonance Energy Transfer / methods Protein Conformation Receptors, G-Protein-Coupled / chemistry

Journal

Nature methods
ISSN: 1548-7105
Titre abrégé: Nat Methods
Pays: United States
ID NLM: 101215604

Informations de publication

Date de publication:
04 2021
Historique:
received: 25 02 2020
accepted: 29 01 2021
pubmed: 10 3 2021
medline: 29 6 2021
entrez: 9 3 2021
Statut: ppublish

Résumé

Class C G protein-coupled receptors (GPCRs) are known to form stable homodimers or heterodimers critical for function, but the oligomeric status of class A and B receptors, which constitute >90% of all GPCRs, remains hotly debated. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful approach with the potential to reveal valuable insights into GPCR organization but has rarely been used in living cells to study protein systems. Here, we report generally applicable methods for using smFRET to detect and track transmembrane proteins diffusing within the plasma membrane of mammalian cells. We leverage this in-cell smFRET approach to show agonist-induced structural dynamics within individual metabotropic glutamate receptor dimers. We apply these methods to representative class A, B and C receptors, finding evidence for receptor monomers, density-dependent dimers and constitutive dimers, respectively.

Identifiants

DOI: 10.1038/s41592-021-01081-y PMID: 33686301 PMC: PMC8232828
pubmed: 33686301
doi: 10.1038/s41592-021-01081-y
pii: 10.1038/s41592-021-01081-y
pmc: PMC8232828
mid: NIHMS1714965
doi:

Substances chimiques

Receptors, G-Protein-Coupled 0

Types de publication

Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S.

Langues

eng

Sous-ensembles de citation

IM

Pagination

397-405

Subventions

Organisme : NIMH NIH HHS
ID : T32 MH018870
Pays : United States
Organisme : NIMH NIH HHS
ID : R01 MH054137
Pays : United States
Organisme : NIGMS NIH HHS
ID : R01 GM098859
Pays : United States
Organisme : NIGMS NIH HHS
ID : R35 GM119619
Pays : United States
Organisme : NEI NIH HHS
ID : R15 EY024451
Pays : United States

Commentaires et corrections

Type : CommentIn

Références

Asher, W. B. et al. Extreme vetting of dopamine receptor oligomerization. In Receptors 33, 99–127 (Humana Press, 2017).
Lambert, N. A. & Javitch, J. A. CrossTalk opposing view: weighing the evidence for class A GPCR dimers, the jury is still out. J. Physiol. 592, 2443–2445 (2014).
pubmed: 24931945 pmcid: 4080927 doi: 10.1113/jphysiol.2014.272997
Doumazane, E. et al. Illuminating the activation mechanisms and allosteric properties of metabotropic glutamate receptors. Proc. Natl Acad. Sci. USA 110, E1416–E1425 (2013).
pubmed: 23487753 pmcid: 3625292 doi: 10.1073/pnas.1215615110
Maurel, D. et al. Cell-surface protein–protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization. Nat. Methods 5, 561–567 (2008).
pubmed: 18488035 pmcid: 2642604 doi: 10.1038/nmeth.1213
White, J. H. et al. Heterodimerization is required for the formation of a functional GABA
pubmed: 9872316 doi: 10.1038/25354
Scarselli, M. et al. Revealing G-protein-coupled receptor oligomerization at the single-molecule level through a nanoscopic lens: methods, dynamics and biological function. FEBS J. 283, 1197–1217 (2016).
pubmed: 26509747 doi: 10.1111/febs.13577
Felce, J. H., Davis, S. J. & Klenerman, D. Single-molecule analysis of G protein-coupled receptor stoichiometry: approaches and limitations. Trends Pharmacol. Sci. 39, 96–108 (2018).
pubmed: 29122289 doi: 10.1016/j.tips.2017.10.005
Kasai, R. S. et al. Full characterization of GPCR monomer–dimer dynamic equilibrium by single molecule imaging. J. Cell Biol. 192, 463–480 (2011).
pubmed: 21300851 pmcid: 3101103 doi: 10.1083/jcb.201009128
Tabor, A. et al. Visualization and ligand-induced modulation of dopamine receptor dimerization at the single molecule level. Sci. Rep. 6, 33233 (2016).
pubmed: 27615810 pmcid: 5018964 doi: 10.1038/srep33233
Calebiro, D. et al. Single-molecule analysis of fluorescently labeled G-protein-coupled receptors reveals complexes with distinct dynamics and organization. Proc. Natl Acad. Sci. USA 110, 743–748 (2013).
pubmed: 23267088 doi: 10.1073/pnas.1205798110
Kasai, R. S., Ito, S. V., Awane, R. M., Fujiwara, T. K. & Kusumi, A. The class-A GPCR dopamine D2 receptor forms transient dimers stabilized by agonists: detection by single-molecule tracking. Cell Biochem. Biophys. 76, 29–37 (2018).
pubmed: 29116599 doi: 10.1007/s12013-017-0829-y
Juette, M. F. et al. The bright future of single-molecule fluorescence imaging. Curr. Opin. Chem. Biol. 20, 103–111 (2014).
pubmed: 24956235 doi: 10.1016/j.cbpa.2014.05.010
Hellenkamp, B. et al. Precision and accuracy of single-molecule FRET measurements—a multi-laboratory benchmark study. Nat. Methods 15, 669–676 (2018).
pubmed: 30171252 pmcid: 6121742 doi: 10.1038/s41592-018-0085-0
Plitzko, J. M., Schuler, B. & Selenko, P. Structural Biology outside the box—inside the cell. Curr. Opin. Struct. Biol. 46, 110–121 (2017).
pubmed: 28735108 doi: 10.1016/j.sbi.2017.06.007
Sustarsic, M. & Kapanidis, A. N. Taking the ruler to the jungle: single-molecule FRET for understanding biomolecular structure and dynamics in live cells. Curr. Opin. Struct. Biol. 34, 52–59 (2015).
pubmed: 26295172 doi: 10.1016/j.sbi.2015.07.001
Sakon, J. J. & Weninger, K. R. Detecting the conformation of individual proteins in live cells. Nat. Methods 7, 203–205 (2010).
pubmed: 20118931 pmcid: 2844853 doi: 10.1038/nmeth.1421
Konig, I. et al. Single-molecule spectroscopy of protein conformational dynamics in live eukaryotic cells. Nat. Methods 12, 773–779 (2015).
pubmed: 26147918 doi: 10.1038/nmeth.3475
Winckler, P. et al. Identification and super-resolution imaging of ligand-activated receptor dimers in live cells. Sci. Rep. 3, 2387 (2013).
pubmed: 23925048 pmcid: 3737505 doi: 10.1038/srep02387
Sako, Y., Minoghchi, S. & Yanagida, T. Single-molecule imaging of EGFR signalling on the surface of living cells. Nat. Cell Biol. 2, 168–172 (2000).
pubmed: 10707088 doi: 10.1038/35004044
Wilmes, S. et al. Mechanism of homodimeric cytokine receptor activation and dysregulation by oncogenic mutations. Science 367, 643–652 (2020).
pubmed: 32029621 pmcid: 8117407 doi: 10.1126/science.aaw3242
Zheng, Q. et al. Electronic tuning of self-healing fluorophores for live-cell and single-molecule imaging. Chem. Sci. 8, 755–762 (2017).
pubmed: 28377799 doi: 10.1039/C6SC02976K
Pati, A. K. et al. Tuning the Baird aromatic triplet-state energy of cyclooctatetraene to maximize the self-healing mechanism in organic fluorophores. Proc. Natl Acad. Sci. USA 117, 24305–24315 (2020).
pubmed: 32913060 pmcid: 7533661 doi: 10.1073/pnas.2006517117
Ishitsuka, Y. et al. Evaluation of genetically encoded chemical tags as orthogonal fluorophore labeling tools for single-molecule FRET applications. J. Phys. Chem. B 119, 6611–6619 (2015).
pubmed: 25978145 doi: 10.1021/acs.jpcb.5b03584
Vafabakhsh, R., Levitz, J. & Isacoff, E. Y. Conformational dynamics of a class C G-protein-coupled receptor. Nature 524, 497–501 (2015).
pubmed: 26258295 pmcid: 4597782 doi: 10.1038/nature14679
Keppler, A. et al. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat. Biotechnol. 21, 86–89 (2003).
pubmed: 12469133 doi: 10.1038/nbt765
Sun, X. et al. Development of SNAP-tag fluorogenic probes for wash-free fluorescence imaging. ChemBioChem 12, 2217–2226 (2011).
pubmed: 21793150 pmcid: 3213346 doi: 10.1002/cbic.201100173
Kawashima, N. et al. Reversible dimerization of EGFR revealed by single-molecule fluorescence imaging using quantum dots. Chemistry 16, 1186–1192 (2010).
pubmed: 20024999 doi: 10.1002/chem.200902963
Suzuki, K. G. et al. Transient GPI-anchored protein homodimers are units for raft organization and function. Nat. Chem. Biol. 8, 774–783 (2012).
pubmed: 22820419 doi: 10.1038/nchembio.1028
Koyama-Honda, I. et al. Fluorescence imaging for monitoring the colocalization of two single molecules in living cells. Biophys. J. 88, 2126–2136 (2005).
pubmed: 15596511 doi: 10.1529/biophysj.104.048967
Jaqaman, K. et al. Robust single-particle tracking in live-cell time-lapse sequences. Nat. Methods 5, 695–702 (2008).
pubmed: 18641657 pmcid: 2747604 doi: 10.1038/nmeth.1237
Vega, A. R., Freeman, S. A., Grinstein, S. & Jaqaman, K. Multistep track segmentation and motion classification for transient mobility analysis. Biophys. J. 114, 1018–1025 (2018).
pubmed: 29539390 pmcid: 5883548 doi: 10.1016/j.bpj.2018.01.012
Gregorio, G. G. et al. Single-molecule analysis of ligand efficacy in β
pubmed: 28607487 pmcid: 5502743 doi: 10.1038/nature22354
Dijkman, P. M. et al. Dynamic tuneable G protein-coupled receptor monomer–dimer populations. Nat. Commun. 9, 1710 (2018).
pubmed: 29703992 pmcid: 5923235 doi: 10.1038/s41467-018-03727-6
Olofsson, L. et al. Fine tuning of sub-millisecond conformational dynamics controls metabotropic glutamate receptors agonist efficacy. Nat. Commun. 5, 5206 (2014).
pubmed: 25323157 doi: 10.1038/ncomms6206
Ferre, S. et al. G protein-coupled receptor oligomerization revisited: functional and pharmacological perspectives. Pharmacol. Rev. 66, 413–434 (2014).
pubmed: 24515647 pmcid: 3973609 doi: 10.1124/pr.113.008052
Meral, D. et al. Molecular details of dimerization kinetics reveal negligible populations of transient micro-opioid receptor homodimers at physiological concentrations. Sci. Rep. 8, 7705 (2018).
pubmed: 29769636 pmcid: 5955887 doi: 10.1038/s41598-018-26070-8
Harikumar, K. G. & Miller, L. J. Secretin receptor dimerization. Prototypic of class B GPCR behavior. In Receptors 33, 273–287 (Humana Press, 2017).
Ward, R. J., Pediani, J. D., Harikumar, K. G., Miller, L. J. & Milligan, G. Spatial intensity distribution analysis quantifies the extent and regulation of homodimerization of the secretin receptor. Biochem. J. 474, 1879–1895 (2017).
pubmed: 28424368 doi: 10.1042/BCJ20170184
El Moustaine, D. et al. Distinct roles of metabotropic glutamate receptor dimerization in agonist activation and G-protein coupling. Proc. Natl Acad. Sci. USA 109, 16342–16347 (2012).
pubmed: 22988116 pmcid: 3479612 doi: 10.1073/pnas.1205838109
Smith, A. W. Detection of rhodopsin dimerization in situ by PIE-FCCS, a time-resolved fluorescence spectroscopy. Methods Mol. Biol. 1271, 205–219 (2015).
pubmed: 25697526 doi: 10.1007/978-1-4939-2330-4_14
Comar, W. D., Schubert, S. M., Jastrzebska, B., Palczewski, K. & Smith, A. W. Time-resolved fluorescence spectroscopy measures clustering and mobility of a G protein-coupled receptor opsin in live cell membranes. J. Am. Chem. Soc. 136, 8342–8349 (2014).
pubmed: 24831851 pmcid: 4063175 doi: 10.1021/ja501948w
Jastrzebska, B. et al. A G protein-coupled receptor dimerization interface in human cone opsins. Biochemistry 56, 61–72 (2017).
pubmed: 28045251 doi: 10.1021/acs.biochem.6b00877
Kaliszewski, M. J. et al. Quantifying membrane protein oligomerization with fluorescence cross-correlation spectroscopy. Methods 140–141, 40–51 (2018).
pubmed: 29448037 pmcid: 5999557 doi: 10.1016/j.ymeth.2018.02.002
Brameshuber, M. & Schutz, G. J. Detection and quantification of biomolecular association in living cells using single-molecule microscopy. Methods Enzymol. 505, 159–186 (2012).
pubmed: 22289453 doi: 10.1016/B978-0-12-388448-0.00017-6
Mudumbi, K. C., Schirmer, E. C. & Yang, W. Single-point single-molecule FRAP distinguishes inner and outer nuclear membrane protein distribution. Nat. Commun. 7, 12562 (2016).
pubmed: 27558844 pmcid: 5007294 doi: 10.1038/ncomms12562
Lacy, M. M., Baddeley, D. & Berro, J. Single-molecule imaging of the BAR-domain protein Pil1p reveals filament-end dynamics. Mol. Biol. Cell 28, 2251–2259 (2017).
pubmed: 28659415 pmcid: 5555653 doi: 10.1091/mbc.e17-04-0238
Zhang, M. et al. Quantitative characterization of the membrane dynamics of newly delivered TGF-β receptors by single-molecule imaging. Anal. Chem. 90, 4282–4287 (2018).
pubmed: 29509006 doi: 10.1021/acs.analchem.7b03448
Zhao, Y. et al. Single-molecule dynamics of gating in a neurotransmitter transporter homologue. Nature 465, 188–193 (2010).
pubmed: 20463731 pmcid: 2940119 doi: 10.1038/nature09057
Juette, M. F. et al. Single-molecule imaging of non-equilibrium molecular ensembles on the millisecond timescale. Nat. Methods 13, 341–344 (2016).
pubmed: 26878382 pmcid: 4814340 doi: 10.1038/nmeth.3769
Elshenawy, M. M. et al. Cargo adaptors regulate stepping and force generation of mammalian dynein–dynactin. Nat. Chem. Biol. 15, 1093–1101 (2019).
pubmed: 31501589 pmcid: 6810841 doi: 10.1038/s41589-019-0352-0
Los, G. V. et al. HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol. 3, 373–382 (2008).
pubmed: 18533659 doi: 10.1021/cb800025k
Hammond, S., Kaplarevic, M., Borth, N., Betenbaugh, M. J. & Lee, K. H. Chinese hamster genome database: an online resource for the CHO community at www.CHOgenome.org. Biotechnol. Bioeng. 109, 1353–1356 (2012).
pubmed: 22105744 doi: 10.1002/bit.24374
O’Gorman, S., Fox, D. T. & Wahl, G. M. Recombinase-mediated gene activation and site-specific integration in mammalian cells. Science 251, 1351–1355 (1991).
pubmed: 1900642 doi: 10.1126/science.1900642
Altman, R. B. et al. Cyanine fluorophore derivatives with enhanced photostability. Nat. Methods 9, 68–71 (2011).
pubmed: 22081126 pmcid: 3433158 doi: 10.1038/nmeth.1774
Tinevez, J. Y. et al. TrackMate: an open and extensible platform for single-particle tracking. Methods 115, 80–90 (2017).
pubmed: 27713081 doi: 10.1016/j.ymeth.2016.09.016
Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).
pubmed: 22930834 pmcid: 5554542 doi: 10.1038/nmeth.2089
Qin, F. Restoration of single-channel currents using the segmental k-means method based on hidden Markov modeling. Biophys. J. 86, 1488–1501 (2004).
pubmed: 14990476 pmcid: 1303984 doi: 10.1016/S0006-3495(04)74217-4

Auteurs

Wesley B Asher (WB)

Department of Psychiatry, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA.
Department of Molecular Pharmacology and Therapeutics, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA.
Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, NY, USA.
ORCID: 0000-0001-7437-218X

Peter Geggier (P)

Department of Psychiatry, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA.
Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, NY, USA.

Michael D Holsey (MD)

Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, NY, USA.
Department of Physiology and Cellular Biophysics, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA.

Grant T Gilmore (GT)

Department of Chemistry, University of Akron, Akron, OH, USA.
ORCID: 0000-0002-0649-8554

Avik K Pati (AK)

Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN, USA.

Jozsef Meszaros (J)

Department of Psychiatry, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA.
Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, NY, USA.

Daniel S Terry (DS)

Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN, USA.

Signe Mathiasen (S)

Department of Psychiatry, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA.
Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, NY, USA.

Megan J Kaliszewski (MJ)

Department of Chemistry, University of Akron, Akron, OH, USA.

Mitchell D McCauley (MD)

Department of Chemistry, University of Akron, Akron, OH, USA.

Alekhya Govindaraju (A)

Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, NY, USA.

Zhou Zhou (Z)

Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY, USA.
Department of Chemistry, Queensborough Community College, The City University of New York, Bayside, NY, USA.
ORCID: 0000-0003-3007-5786

Kaleeckal G Harikumar (KG)

Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, AZ, USA.

Khuloud Jaqaman (K)

Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Lyda Hill Department of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, TX, USA.

Laurence J Miller (LJ)

Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, AZ, USA.

Adam W Smith (AW)

Department of Chemistry, University of Akron, Akron, OH, USA.

Scott C Blanchard (SC)

Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN, USA. scott.blanchard@stjude.org.
Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY, USA. scott.blanchard@stjude.org.
ORCID: 0000-0003-2717-9365

Jonathan A Javitch (JA)

Department of Psychiatry, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA. jonathan.javitch@nyspi.columbia.edu.
Department of Molecular Pharmacology and Therapeutics, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA. jonathan.javitch@nyspi.columbia.edu.
Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, NY, USA. jonathan.javitch@nyspi.columbia.edu.
Department of Physiology and Cellular Biophysics, Vagelos College of Physicians and Surgeons, Columbia University, New York, NY, USA. jonathan.javitch@nyspi.columbia.edu.
ORCID: 0000-0001-7395-2967

Articles similaires

Structural basis for molecular assembly of fucoxanthin chlorophyll

Koji Kato, Yoshiki Nakajima, Jian Xing et al.
1.00
Diatoms Photosystem I Protein Complex Chlorophyll Binding Proteins Cryoelectron Microscopy Light-Harvesting Protein Complexes

The conformational landscape of fold-switcher KaiB is tuned to the circadian rhythm timescale.

Hannah K Wayment-Steele, Renee Otten, Warintra Pitsawong et al.
1.00
Rhodobacter sphaeroides Bacterial Proteins Circadian Rhythm Protein Folding Protein Conformation

Catalytic peptide-based coacervates for enhanced function through structural organization and substrate specificity.

David Q P Reis, Sara Pereira, Ana P Ramos et al.
1.00
Substrate Specificity Peptides Catalysis Hydrolysis Protein Conformation

Structural dynamics of human deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase).

Ravdna Sarre, Olena Dobrovolska, Patrik Lundström et al.
1.00
Humans Pyrophosphatases Protein Conformation Molecular Dynamics Simulation Kinetics

Classifications MeSH

questionsmedicales.fr › Métabolisme › Dimérisation › Single-molecule FRET imaging of GPCR dimers in...
questionsmedicales.fr › Phénomènes chimiques › Phénomènes biochimiques › Transfert d'énergie par résonance de fluorescence › Single-molecule FRET imaging of GPCR dimers in...
questionsmedicales.fr › Phénomènes chimiques › Phénomènes biochimiques › Conformation moléculaire › Conformation des protéines › Single-molecule FRET imaging of GPCR dimers in...
questionsmedicales.fr › Acides aminés, peptides et protéines › Protéines › Protéines membranaires › Récepteurs de surface cellulaire › Récepteurs couplés aux protéines G › Single-molecule FRET imaging of GPCR dimers in...