Optical Probing of Metabotropic Glutamate Receptors

Abstract

G protein-coupled receptors (GPCRs), the largest family of membrane proteins, enable cells to respond to a wide array of extracellular stimuli and control a diverse array of intracellular signaling pathways. The Class C GPCRs, which contain the metabotropic glutamate receptors (mGluRs) and GABAB receptors, are expressed throughout the nervous system where they control excitability and synaptic strength and serve as drug targets for many neurological disorders. Despite the presence of high resolution crystal structures of isolated domains, many questions remain about the dynamic processes of receptor activation and G protein coupling in full length receptors. Furthermore, the limitations of pharmacology and classical assays have made it difficult to control and measure individual receptors with high spatiotemporal precision and subtype specificity. In this study we have developed and refined methods for attaching photoswitchable tethered ligands (PTLs) for the in vivo photocontrol of mGluRs, with a focus on mGluR2. Optical control of mGluR2 via tethering of photoswitches to SNAP-tags allowed for efficient, orthogonal labeling of mGluR2 without the complications of maleimide chemistry. This new approach to PTL tethering allowed for dual optical control of multiple receptors within the same cell. We also used PTLs to selectively ligand one or both memebers of linked mGluR2 homodimers to reveal substantial cooperativity. Strikingly, in mGluR2/3 heterodimers this cooperativity is asymmetric. These experiments were complemented with intersubunit ensemble and single molecule fluorescence resonance energy transfer (FRET) experiments that revealed the role of the dimer interface in the conformational dynamics underling receptor activation. Together this work contributes a number of new tools for optical probing of GPCRs and leads to a model for subunit-dependent activation of mGluRs.