1. Single-molecule FRET imaging of GPCR dimers in living cells
- Author
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Michael D. Holsey, Grant T. Gilmore, Adam W. Smith, Jonathan A. Javitch, Avik Kumar Pati, Kaleeckal G. Harikumar, Zhou Zhou, Jozsef Meszaros, Laurence J. Miller, Khuloud Jaqaman, Peter Geggier, Signe Mathiasen, Wesley B. Asher, Megan J. Kaliszewski, Mitchell D. McCauley, Scott C. Blanchard, Daniel S. Terry, and Alekhya Govindaraju
- Subjects
0303 health sciences ,Chemistry ,Cell Biology ,Single-molecule FRET ,Biochemistry ,Transmembrane protein ,03 medical and health sciences ,Protein structure ,Förster resonance energy transfer ,Metabotropic glutamate receptor ,Biophysics ,Receptor ,Molecular Biology ,Function (biology) ,030304 developmental biology ,Biotechnology ,G protein-coupled receptor - 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.
- Published
- 2021