Single-molecule FRET Imaging of GPCR Dimers in Living Cells

Overview

Journal Nat Methods

Specialties Biomedical Engineering
Pathology

Date 2021 Mar 9

PMID 33686301

Citations 71

Authors

Wesley B Asher

Peter Geggier

Michael D Holsey

Grant T Gilmore

Avik K Pati

Jozsef Meszaros

Daniel S Terry

Signe Mathiasen

Megan J Kaliszewski

Mitchell D McCauley

Alekhya Govindaraju

Zhou Zhou

Kaleeckal G Harikumar

Khuloud Jaqaman

Laurence J Miller

Adam W Smith

Scott C Blanchard

Jonathan A Javitch

Affiliations

Soon will be listed here.

Abstract

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.

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References

Lambert N, Javitch J . CrossTalk opposing view: Weighing the evidence for class A GPCR dimers, the jury is still out. J Physiol. 2014; 592(12):2443-5. PMC: 4080927. DOI: 10.1113/jphysiol.2014.272997. View

Doumazane E, Scholler P, Fabre L, Zwier J, Trinquet E, Pin J . Illuminating the activation mechanisms and allosteric properties of metabotropic glutamate receptors. Proc Natl Acad Sci U S A. 2013; 110(15):E1416-25. PMC: 3625292. DOI: 10.1073/pnas.1215615110. View

Maurel D, Comps-Agrar L, Brock C, Rives M, Bourrier E, Ayoub M . Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization. Nat Methods. 2008; 5(6):561-7. PMC: 2642604. DOI: 10.1038/nmeth.1213. View

White J, Wise A, Main M, Green A, Fraser N, Disney G . Heterodimerization is required for the formation of a functional GABA(B) receptor. Nature. 1999; 396(6712):679-82. DOI: 10.1038/25354. View

Scarselli M, Annibale P, McCormick P, Kolachalam S, Aringhieri S, Radenovic A . Revealing G-protein-coupled receptor oligomerization at the single-molecule level through a nanoscopic lens: methods, dynamics and biological function. FEBS J. 2015; 283(7):1197-217. DOI: 10.1111/febs.13577. View

Felce J, Davis S, Klenerman D . Single-Molecule Analysis of G Protein-Coupled Receptor Stoichiometry: Approaches and Limitations. Trends Pharmacol Sci. 2017; 39(2):96-108. DOI: 10.1016/j.tips.2017.10.005. View

Kasai R, Suzuki K, Prossnitz E, Koyama-Honda I, Nakada C, Fujiwara T . Full characterization of GPCR monomer-dimer dynamic equilibrium by single molecule imaging. J Cell Biol. 2011; 192(3):463-80. PMC: 3101103. DOI: 10.1083/jcb.201009128. View

Tabor A, Weisenburger S, Banerjee A, Purkayastha N, Kaindl J, Hubner H . Visualization and ligand-induced modulation of dopamine receptor dimerization at the single molecule level. Sci Rep. 2016; 6:33233. PMC: 5018964. DOI: 10.1038/srep33233. View

Calebiro D, Rieken F, Wagner J, Sungkaworn T, Zabel U, Borzi A . Single-molecule analysis of fluorescently labeled G-protein-coupled receptors reveals complexes with distinct dynamics and organization. Proc Natl Acad Sci U S A. 2012; 110(2):743-8. PMC: 3545784. DOI: 10.1073/pnas.1205798110. View

10.

Kasai R, Ito S, Awane R, Fujiwara T, Kusumi A . The Class-A GPCR Dopamine D2 Receptor Forms Transient Dimers Stabilized by Agonists: Detection by Single-Molecule Tracking. Cell Biochem Biophys. 2017; 76(1-2):29-37. PMC: 5913388. DOI: 10.1007/s12013-017-0829-y. View

11.

Juette M, Terry D, Wasserman M, Zhou Z, Altman R, Zheng Q . The bright future of single-molecule fluorescence imaging. Curr Opin Chem Biol. 2014; 20:103-11. PMC: 4123530. DOI: 10.1016/j.cbpa.2014.05.010. View

12.

Hellenkamp B, Schmid S, Doroshenko O, Opanasyuk O, Kuhnemuth R, Rezaei Adariani S . Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study. Nat Methods. 2018; 15(9):669-676. PMC: 6121742. DOI: 10.1038/s41592-018-0085-0. View

13.

Plitzko J, Schuler B, Selenko P . Structural Biology outside the box-inside the cell. Curr Opin Struct Biol. 2017; 46:110-121. DOI: 10.1016/j.sbi.2017.06.007. View

14.

Sustarsic M, Kapanidis A . Taking the ruler to the jungle: single-molecule FRET for understanding biomolecular structure and dynamics in live cells. Curr Opin Struct Biol. 2015; 34:52-9. DOI: 10.1016/j.sbi.2015.07.001. View

15.

Sakon J, Weninger K . Detecting the conformation of individual proteins in live cells. Nat Methods. 2010; 7(3):203-5. PMC: 2844853. DOI: 10.1038/nmeth.1421. View

16.

Konig I, Zarrine-Afsar A, Aznauryan M, Soranno A, Wunderlich B, Dingfelder F . Single-molecule spectroscopy of protein conformational dynamics in live eukaryotic cells. Nat Methods. 2015; 12(8):773-9. DOI: 10.1038/nmeth.3475. View

17.

Winckler P, Lartigue L, Giannone G, De Giorgi F, Ichas F, Sibarita J . Identification and super-resolution imaging of ligand-activated receptor dimers in live cells. Sci Rep. 2013; 3:2387. PMC: 3737505. DOI: 10.1038/srep02387. View

18.

Sako Y, Minoghchi S, Yanagida T . Single-molecule imaging of EGFR signalling on the surface of living cells. Nat Cell Biol. 2000; 2(3):168-72. DOI: 10.1038/35004044. View

19.

Wilmes S, Hafer M, Vuorio J, Tucker J, Winkelmann H, Lochte S . Mechanism of homodimeric cytokine receptor activation and dysregulation by oncogenic mutations. Science. 2020; 367(6478):643-652. PMC: 8117407. DOI: 10.1126/science.aaw3242. View

20.

Zheng Q, Jockusch S, Zhou Z, Altman R, Zhao H, Asher W . Electronic tuning of self-healing fluorophores for live-cell and single-molecule imaging. Chem Sci. 2017; 8(1):755-762. PMC: 5299821. DOI: 10.1039/C6SC02976K. View