Abreu, N. | Search bioRxiv

bioRxiv2023

Structural basis of allosteric modulation ofmetabotropic glutamate receptor activation and desensitization

The metabotropic glutamate receptors (mGluRs) are neuromodulatory family C G protein coupled receptors which assemble as dimers and allosterically couple extracellular ligand binding domains (LBDs) to transmembrane domains (TMDs) to drive intracellular signaling. Pharmacologically, mGluRs can be targeted either at the LBDs by glutamate and synthetic orthosteric compounds or at the TMDs by allosteric modulators. Despite the potential of allosteric TMD-targeting compounds as therapeutics, an understanding of the functional and structural basis of their effects on mGluRs is limited. Here we use a battery of approaches to dissect the distinct functional and structural effects of orthosteric versus allosteric ligands. We find using electrophysiological and live cell imaging assays that both agonists and positive allosteric modulators (PAMs) can drive activation and desensitization of mGluRs. The effects of PAMs are pleiotropic, including both the ability to boost the maximal response to orthosteric agonists and to serve independently as desensitization-biased agonists across mGluR subtypes. Conformational sensors reveal PAM-driven inter-subunit re-arrangements at both the LBD and TMD. Motivated by this, we determine cryo-electron microscopy structures of mGluR3 in the presence of either an agonist or antagonist alone or in combination with a PAM. These structures reveal PAM-driven re-shaping of intra- and inter-subunit conformations and provide evidence for a rolling TMD dimer interface activation pathway that controls G protein and beta-arrestin coupling. Highlights-Agonists and PAMs drive mGluR activation, desensitization, and endocytosis -PAMs are desensitization-biased and synergistic with agonists -Four combinatorial ligand conditions reveal an ensemble of full-length mGluR structures with novel interfaces -Activation and desensitization involve rolling TMD interfaces which are re-shaped by PAM

biophysics↗

bioRxiv2022

Profiling the diversity of agonist-selective effects on the proximal proteome environment of G protein-coupled receptors

The mu opioid receptor (OR), a prototypic member of the large G protein-coupled receptor (GPCR) family, represents an important target of therapeutic and abused drugs. To date, most of our understanding of OR activity has focused on signal transducers and regulatory molecules including G proteins, GPCR kinases, and beta-arrestins. Yet it is clear that signaling through the OR is coordinated by additional proteins recruited into the proximal interaction network of the activated receptor, which have largely remained invisible given the lack of technologies to interrogate these networks systematically. Here, we implement a quantitative proteomics pipeline leveraging the chemical diversity of OR agonists and APEX-based proximity labeling to investigate the protein networks that underlie OR signaling. We leverage a novel computational framework to extract subcellular location, trafficking, and functional partners of GPCR activity from the proximity labeling datasets. Applying this unbiased, systematic approach to the OR, we demonstrate that opioid agonists exert differences in the OR proximal proteome mediated by endocytosis and subsequent endosomal sorting, exemplified by VPS35 and COMMD3. Moreover, we identify two novel OR network components, EYA4 and KCTD12, that are recruited into the receptor proximal network irrespective of the activating ligand and independent of receptor trafficking but based on receptor-triggered G protein activation. We provide functional evidence that these network components form a previously unrecognized buffering system for G protein activity which broadly modulates cellular GPCR signaling.

systems biology↗