Single-molecule FRET imaging of GPCR dimers in living cells

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

doi:10.1038/s41592-021-01081-y

Single-molecule FRET imaging of GPCR dimers in living cells

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

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97 Scopus citations

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.

Original language	English (US)
Pages (from-to)	397-405
Number of pages	9
Journal	Nature methods
Volume	18
Issue number	4
DOIs	https://doi.org/10.1038/s41592-021-01081-y
State	Published - Apr 2021

ASJC Scopus subject areas

Biotechnology
Biochemistry
Molecular Biology
Cell Biology

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Asher, W. B., Geggier, P., Holsey, M. D., Gilmore, G. T., Pati, A. K., Meszaros, J., Terry, D. S., Mathiasen, S., Kaliszewski, M. J., McCauley, M. D., Govindaraju, A., Zhou, Z., Harikumar, K. G., Jaqaman, K., Miller, L. J., Smith, A. W., Blanchard, S. C., & Javitch, J. A. (2021). Single-molecule FRET imaging of GPCR dimers in living cells. Nature methods, 18(4), 397-405. https://doi.org/10.1038/s41592-021-01081-y

Asher, WB, Geggier, P, Holsey, MD, Gilmore, GT, Pati, AK, Meszaros, J, Terry, DS, Mathiasen, S, Kaliszewski, MJ, McCauley, MD, Govindaraju, A, Zhou, Z, Harikumar, KG, Jaqaman, K, Miller, LJ, Smith, AW, Blanchard, SC & Javitch, JA 2021, 'Single-molecule FRET imaging of GPCR dimers in living cells', Nature methods, vol. 18, no. 4, pp. 397-405. https://doi.org/10.1038/s41592-021-01081-y

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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.",

author = "Asher, {Wesley B.} and Peter Geggier and Holsey, {Michael D.} and Gilmore, {Grant T.} and Pati, {Avik K.} and Jozsef Meszaros and Terry, {Daniel S.} and Signe Mathiasen and Kaliszewski, {Megan J.} and McCauley, {Mitchell D.} and Alekhya Govindaraju and Zhou Zhou and Harikumar, {Kaleeckal G.} and Khuloud Jaqaman and Miller, {Laurence J.} and Smith, {Adam W.} and Blanchard, {Scott C.} and Javitch, {Jonathan A.}",

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AU - Geggier, Peter

AU - Holsey, Michael D.

AU - Gilmore, Grant T.

AU - Pati, Avik K.

AU - Meszaros, Jozsef

AU - Terry, Daniel S.

AU - Mathiasen, Signe

AU - Kaliszewski, Megan J.

AU - McCauley, Mitchell D.

AU - Govindaraju, Alekhya

AU - Zhou, Zhou

AU - Harikumar, Kaleeckal G.

AU - Jaqaman, Khuloud

AU - Miller, Laurence J.

AU - Smith, Adam W.

AU - Blanchard, Scott C.

AU - Javitch, Jonathan A.

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Single-molecule FRET imaging of GPCR dimers in living cells

Abstract

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