The Activation Dynamics of Class C Gpcrs Revealed by Single Molecule FRET

Abstract

G-protein-coupled receptors (GPCRs) are the largest and most diverse family of membrane signaling proteins. Binding of an extracellular signal to a GPCR activates G protein, which in turn modulates the activity of different downstream cellular effectors. Recent biochemical, pharmacological and biophysical studies suggest that GPCRs do not simply act as on-off switches but instead exist in multiple conformational and functional states where they may signal to distinct G proteins and other partners. However, despite recent advances, many aspects of signal transduction through GPCRs is still not well understood. Here we report a novel functional assay based on single molecule FRET to probe real-time conformational dynamics of isolated receptors. Using our assay, we studied metabotropic glutamate receptors (mGluRs), dimeric members of class C GPCR family, which are involved in modulation of synaptic transmission and excitability of nerve cells. Experiments on mGluR2 and mGluR3 homodimers reveal multiple stable conformational states. Strikingly, in spite of more than 70% sequence identity between mGluR2 and mGluR3, we observe substantial differences in their inherent dynamics and basal activity in absence of ligand. Our approach should make it possible to directly quantify how drugs stabilize conformational states of receptors and provide new insights into the molecular mechanisms of GPCR signaling.