Profiling the proximal proteome of the activated µ-opioid receptor.

The µ-opioid receptor (µOR) represents an important target of therapeutic and abused drugs. So far, most understanding of µOR activity has focused on a subset of known signal transducers and regulatory molecules. Yet µOR signaling is coordinated by additional proteins in the interaction network of the activated receptor, which have largely remained invisible given the lack of technologies to interrogate these networks systematically. Here we describe a proteomics and computational approach to map the proximal proteome of the activated µOR and to extract subcellular location, trafficking and functional partners of G-protein-coupled receptor (GPCR) activity. We demonstrate that distinct opioid agonists exert differences in the µOR proximal proteome mediated by endocytosis and endosomal sorting. Moreover, we identify two new µOR network components, EYA4 and KCTD12, which are recruited on the basis of receptor-triggered G-protein activation and might form a previously unrecognized buffering system for G-protein activity broadly modulating cellular GPCR signaling.

An epigenetic barrier sets the timing of human neuronal maturation.

The pace of human brain development is highly protracted compared with most other species1-7. The maturation of cortical neurons is particularly slow, taking months to years to develop adult functions3-5. Remarkably, such protracted timing is retained in cortical neurons derived from human pluripotent stem cells (hPSCs) during in vitro differentiation or upon transplantation into the mouse brain4,8,9. Those findings suggest the presence of a cell-intrinsic clock setting the pace of neuronal maturation, although the molecular nature of this clock remains unknown. Here we identify an epigenetic developmental programme that sets the timing of human neuronal maturation. First, we developed a hPSC-based approach to synchronize the birth of cortical neurons in vitro which enabled us to define an atlas of morphological, functional and molecular maturation. We observed a slow unfolding of maturation programmes, limited by the retention of specific epigenetic factors. Loss of function of several of those factors in cortical neurons enables precocious maturation. Transient inhibition of EZH2, EHMT1 and EHMT2 or DOT1L, at progenitor stage primes newly born neurons to rapidly acquire mature properties upon differentiation. Thus our findings reveal that the rate at which human neurons mature is set well before neurogenesis through the establishment of an epigenetic barrier in progenitor cells. Mechanistically, this barrier holds transcriptional maturation programmes in a poised state that is gradually released to ensure the prolonged timeline of human cortical neuron maturation.

Dissecting the molecular basis for the modulation of neurotransmitter GPCR signaling by GINIP.

It is well established that G-protein-coupled receptors (GPCRs) stimulated by neurotransmitters are critical for neuromodulation. Much less is known about how heterotrimeric G-protein (Gαßγ) regulation after receptor-mediated activation contributes to neuromodulation. Recent evidence indicates that the neuronal protein GINIP shapes GPCR inhibitory neuromodulation via a unique mechanism of G-protein regulation that controls pain and seizure susceptibility. However, the molecular basis of this mechanism remains ill-defined because the structural determinants of GINIP responsible for binding and regulating G proteins are not known. Here, we combined hydrogen-deuterium exchange mass spectrometry, computational structure predictions, biochemistry, and cell-based biophysical assays to demonstrate an effector-like binding mode of GINIP to Gαi. Specific amino acids of GINIP's PHD domain first loop are essential for G-protein binding and subsequent regulation of Gαi-GTP and Gßγ signaling upon neurotransmitter GPCR stimulation. In summary, these findings shed light onto the molecular basis for a post-receptor mechanism of G-protein regulation that fine-tunes inhibitory neuromodulation.

Distinct beta-arrestin coupling and intracellular trafficking of metabotropic glutamate receptor homo- and heterodimers.

The metabotropic glutamate receptors (mGluRs) are family C, dimeric G protein-coupled receptors (GPCRs), which play critical roles in synaptic transmission. Despite an increasing appreciation of the molecular diversity of this family, how distinct mGluR subtypes are regulated remains poorly understood. We reveal that different group II/III mGluR subtypes show markedly different beta-arrestin (ß-arr) coupling and endocytic trafficking. While mGluR2 is resistant to internalization and mGluR3 shows transient ß-arr coupling, which enables endocytosis and recycling, mGluR8 and ß-arr form stable complexes, which leads to efficient lysosomal targeting and degradation. Using chimeras and mutagenesis, we pinpoint carboxyl-terminal domain regions that control ß-arr coupling and trafficking, including the identification of an mGluR8 splice variant with impaired internalization. We then use a battery of high-resolution fluorescence assays to find that heterodimerization further expands the diversity of mGluR regulation. Together, this work provides insight into the relationship between GPCR/ß-arr complex formation and trafficking while revealing diversity and intricacy in the regulation of mGluRs.

Structural basis of allosteric modulation of metabotropic 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.

Control of G protein-coupled receptor function via membrane-interacting intrinsically disordered C-terminal domains.

G protein-coupled receptors (GPCRs) control intracellular signaling cascades via agonist-dependent coupling to intracellular transducers including heterotrimeric G proteins, GPCR kinases (GRKs), and arrestins. In addition to their critical interactions with the transmembrane core of active GPCRs, all three classes of transducers have also been reported to interact with receptor C-terminal domains (CTDs). An underexplored aspect of GPCR CTDs is their possible role as lipid sensors given their proximity to the membrane. CTD-membrane interactions have the potential to control the accessibility of key regulatory CTD residues to downstream effectors and transducers. Here we report that the CTDs of two closely related family C GPCRs, metabotropic glutamate receptor 2 (mGluR2) and mGluR3, bind to membranes and that this interaction controls receptor function. We first characterize CTD structure with NMR spectroscopy, revealing lipid composition-dependent modes of membrane binding. Using molecular dynamics simulations and structure-guided mutagenesis, we identify key conserved residues and cancer-associated mutations that control CTD-membrane binding. Finally, we provide evidence that mGluR3 transducer coupling is controlled by CTD-membrane interactions in live cells which can be modulated by disease-associated mutations or CTD phosphorylation. This work reveals a novel mechanism of GPCR modulation, suggesting that CTD-membrane binding may be a general regulatory mode throughout the broad GPCR superfamily.

NMDA receptor-BK channel coupling regulates synaptic plasticity in the barrel cortex.

Postsynaptic N-methyl-D-aspartate receptors (NMDARs) are crucial mediators of synaptic plasticity due to their ability to act as coincidence detectors of presynaptic and postsynaptic neuronal activity. However, NMDARs exist within the molecular context of a variety of postsynaptic signaling proteins, which can fine-tune their function. Here, we describe a form of NMDAR suppression by large-conductance Ca2+- and voltage-gated K+ (BK) channels in the basal dendrites of a subset of barrel cortex layer 5 pyramidal neurons. We show that NMDAR activation increases intracellular Ca2+ in the vicinity of BK channels, thus activating K+ efflux and strong negative feedback inhibition. We further show that neurons exhibiting such NMDAR-BK coupling serve as high-pass filters for incoming synaptic inputs, precluding the induction of spike timing-dependent plasticity. Together, these data suggest that NMDAR-localized BK channels regulate synaptic integration and provide input-specific synaptic diversity to a thalamocortical circuit.

Functional validation of Ca2+-binding residues from the crystal structure of the BK ion channel.

BK channels are dually regulated by voltage and Ca2+, providing a cellular mechanism to couple electrical and chemical signalling. Intracellular Ca2+ concentration is sensed by a large cytoplasmic region in the channel known as "gating ring", which is formed by four tandems of regulator of conductance for K+ (RCK1 and RCK2) domains. The recent crystal structure of the full-length BK channel from Aplysia californica has provided new information about the residues involved in Ca2+ coordination at the high-affinity binding sites located in the RCK1 and RCK2 domains, as well as their cooperativity. Some of these residues have not been previously studied in the human BK channel. In this work we have investigated, through site directed mutagenesis and electrophysiology, the effects of these residues on channel activation by voltage and Ca2+. Our results demonstrate that the side chains of two non-conserved residues proposed to coordinate Ca2+ in the A. californica structure (G523 and E591) have no apparent functional role in the human BK Ca2+ sensing mechanism. Consistent with the crystal structure, our data indicate that in the human channel the conserved residue R514 participates in Ca2+ coordination in the RCK1 binding site. Additionally, this study provides functional evidence indicating that R514 also interacts with residues E902 and Y904 connected to the Ca2+ binding site in RCK2. Interestingly, it has been proposed that this interaction may constitute a structural correlate underlying the cooperative interactions between the two high-affinity Ca2+ binding sites regulating the Ca2+ dependent gating of the BK channel. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.