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^*

^*Corresponding author af dette arbejde

Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

30 Citationer (Scopus)

20 Downloads (Pure)

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.

Originalsprog	Engelsk
Tidsskrift	Nature Methods
Vol/bind	18
Udgave nummer	4
Sider (fra-til)	397-405
Antal sider	9
ISSN	1548-7091
DOI	https://doi.org/10.1038/s41592-021-01081-y
Status	Udgivet - 2021
Udgivet eksternt	Ja

Bibliografisk note

Publisher Copyright:
© 2021, The Author(s), under exclusive licence to Springer Nature America, Inc.

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10.1038/s41592-021-01081-y

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Citationsformater

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

Single-molecule FRET imaging of GPCR dimers in living cells. / Asher, Wesley B.; Geggier, Peter; Holsey, Michael D.; Gilmore, Grant T.; Pati, Avik K.; Meszaros, Jozsef; Terry, Daniel S.; Mathiasen, Signe; Kaliszewski, Megan J.; McCauley, Mitchell D.; Govindaraju, Alekhya; Zhou, Zhou; Harikumar, Kaleeckal G.; Jaqaman, Khuloud; Miller, Laurence J; Smith, Adam W.; Blanchard, Scott C.; Javitch, Jonathan A.

I: Nature Methods, Bind 18, Nr. 4, 2021, s. 397-405.

Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

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, bind 18, nr. 4, s. 397-405. https://doi.org/10.1038/s41592-021-01081-y

Asher, Wesley B. ; Geggier, Peter ; Holsey, Michael D. ; Gilmore, Grant T. ; Pati, Avik K. ; Meszaros, Jozsef ; Terry, Daniel S. ; Mathiasen, Signe ; Kaliszewski, Megan J. ; McCauley, Mitchell D. ; Govindaraju, Alekhya ; Zhou, Zhou ; Harikumar, Kaleeckal G. ; Jaqaman, Khuloud ; Miller, Laurence J ; Smith, Adam W. ; Blanchard, Scott C. ; Javitch, Jonathan A. / Single-molecule FRET imaging of GPCR dimers in living cells. I: Nature Methods. 2021 ; Bind 18, Nr. 4. s. 397-405.

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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.",

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AU - Asher, Wesley B.

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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N2 - 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.

AB - 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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