Conformational Rearrangement During Activation of a Metabotropic Glutamate Receptor

Abstract

The ability to sense external stimuli and dynamically respond lies at the core of cellular homeostasis. G protein-coupled receptors (GPCRs), the largest family of membrane proteins in humans, are key components of cellular signal processing. GPCRs are highly dynamic allosteric proteins that function by relaying ligand-induced conformational changes at the extracellular interface to the intracellular G protein-coupling region. Therefore, quantifying GPCR conformational dynamics at key functional domains is essential for understanding their mechanisms of activation and modulation. Among all GPCRs, metabotropic glutamate receptors (mGluRs) are unique as they function as constitutive dimers and possess large extracellular domains housing the ligand binding sites. This unique architecture requires ligand-induced conformational changes to propagate over 120Å. Despite recent structures and microscopy studies, our understanding of how conformational dynamics is relayed through mGluRs is still incomplete. Here, we used a combination of single molecule Förster resonance energy transfer (smFRET) and cell-based fluorescence microscopy to quantify the conformational dynamics of mGluR2. Specifically, we examined the cysteine-rich domains (CRDs) which serve as the allosteric linkers between the extracellular ligand binding domains (LBDs) and the membrane bound heptahelical domains. First, we show that the CRDs of mGluR2 are in dynamic equilibrium among four distinct conformations, two of which were previously unknown, and that transitions between the conformations occur in an ordered and sequential manner. Second, we found that the conformational dynamics of the LBDs and CRDs differ dramatically under the same conditions, serving as direct evidence for loose coupling in mGluRs. Lastly, using smFRET microscopy and mutagenesis, we show that the newly identified intermediate states act as critical conformational checkpoints for regulating mGluR2 activation. Overall, our findings provide a dynamic framework for the activation process of mGluRs specifically and class C GPCRs in general.