Dynamic Mechanisms of Metabotropic Glutamate Receptor Activation and Modulation

Liauw, Brandon Wey-Hung

doi:https://doi.org/10.21985/n2-akjy-9108

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Dynamic Mechanisms of Metabotropic Glutamate Receptor Activation and Modulation

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G protein-coupled receptors (GPCRs) are the largest family of membrane receptors in humans and play a role in nearly all physiological processes. Among GPCRs, metabotropic glutamate receptors (mGluRs) are a structurally distinct family of synaptic receptors that are essential in regulating neurotransmission and synaptic plasticity. Due to their important regulatory role in the central nervous system, mGluRs have emerged as promising targets for treating a range of neurological and psychiatric disorders. The overall objective of this thesis dissertation is to characterize the mechanisms of mGluR activation and modulation.mGluRs function as constitutive dimers and possess large extracellular domains composed of a ligand binding Venus flytrap (VFT) domain and a linker cysteine-rich domain (CRD). In addition, the highly conserved 7 transmembrane (7TM) domain forms an allosteric pocket that can be targeted by synthetic modulators. Thus, mGluR function is dependent on a complex and coordinated transmission of conformational dynamics among three structural domains and two ligand-binding sites. At the beginning of this thesis work, our understanding of mGluR conformational dynamics was fragmented and incomplete. Moreover, how the spatially separated domains and ligand binding sites were conformationally coupled was unclear. Here, we used a combination of single-molecule fluorescence resonance energy transfer (smFRET), cell-based FRET, and functional calcium imaging to quantify the conformational dynamics underlying mGluR2 activation and modulation. During mGluR2 activation, we show that CRDs 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. The intermediate states were identified to act as critical checkpoints for receptor activation. Finally, comparing the VFT domain rearrangement during activation with that of the CRD and 7TM domain revealed dramatically different structural dynamics, providing direct evidence for loose coupling in mGluRs Our four-state activation model provided us with a framework to interpret and describe the effects of both synthetic and endogenous allosteric modulators on mGluR2 conformational dynamics. We identified novel mechanisms for positive and negative allosteric modulation that are dependent on the intrinsic dynamics of the CRD and the two intermediate states. Furthermore, examination of the VFT domain, CRD, and 7TM domain demonstrated that the allosteric modulators tested affect the overall dimeric rearrangement of mGluR2. Lastly, we show that solubilizing agents strongly influence the structural and dynamic properties of mGluRs, emphasizing the importance in optimizing components of the hydrophobic scaffold. Collectively, the findings presented in this thesis established and refined a dynamic framework for mGluR2 activation and modulation that likely extends to other mGluRs and other dimeric multidomain membrane receptors.

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