Single-molecule FRET imaging of GPCR dimers in living cells

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.

Automated summary

This study presents a generally applicable method for using single-molecule fluorescence resonance energy transfer (smFRET) to detect and track transmembrane proteins diffusing within the plasma membrane of mammalian cells. The in-cell smFRET approach reveals agonist-induced structural dynamics within individual metabotropic glutamate receptor dimers. Evidence for receptor monomers, density-dependent dimers, and constitutive dimers in representative class A, B, and C receptors were observed using these methods.