Open AccessSingle-molecule FRET imaging of GPCR dimers in living cells
Author(s) -
Wesley B. Asher,
Peter Geggier,
Michael D. Holsey,
Grant T. Gilmore,
Avik Kumar Pati,
József Mészáros,
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 Javitch
Publication year - 2021
Publication title -
nature methods
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 19.469
H-Index - 318
eISSN - 1548-7105
pISSN - 1548-7091
DOI - 10.1038/s41592-021-01081-y
Subject(s) - g protein coupled receptor , förster resonance energy transfer , single molecule fret , receptor , biophysics , biology , agonist , computational biology , microbiology and biotechnology , chemistry , biochemistry , fluorescence , physics , quantum mechanics
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.