Biology subjects

Gonzalez-Hernandez, A. J.

Publications and source records attributed to Gonzalez-Hernandez, A. J..

6 recordsLinked to original sources

bioRxiv2025

Structural Diversity of Metabotropic Glutamate Receptor/Beta-Arrestin Coupling

Beta-arrestins ({beta}-arrs) are cytosolic proteins which mediate G protein-coupled receptor (GPCR) desensitization, endocytosis, and signaling. Despite the widespread physiological roles of {beta}-arr coupling, the molecular basis of GPCR/{beta}-arr interaction has been studied primarily in monomeric family A GPCRs. Here we take an integrative biophysical and structural approach to uncover molecular diversity in {beta}-arr coupling to the neuromodulatory metabotropic glutamate receptors (mGluRs), prototypical, dimeric family C GPCRs. We find, using a new single molecule pulldown assay, that mGluRs couple to {beta}-arrs with a 2:1 or 2:2 stoichiometry via a combination of "tail" and "core" interactions. Using single molecule FRET analysis, we also find that {beta}-arr1 stabilizes active conformations of mGluR8. Cryo-EM structures of mGluR8 alone or with either G proteins or {beta}-arr1 reveal transducer-specific mGluR8 active states and, in combination with molecular dynamics simulations, define the positioning of mGluR8-bound {beta}-arr1, supporting a steric mechanism of mGluR desensitization involving interactions with both subunits and the lipid bilayer. Finally, combinatorial mutagenesis enables the identification of a landscape of homo- and hetero-dimeric mGluR/{beta}-arr complexes, including mGluR/{beta}-arr1/{beta}-arr2 megacomplexes, providing a framework for family C GPCR/{beta}-arr coupling and expanding the known range of GPCR/transducer coupling modes.

biophysics↗

bioRxiv2023

Deuteration provides a general strategy to enhance azobenzene-based photopharmacology

Herein, we present deuterated azobenzene photoswitches as a general means of enhancing photopharmacological molecules. Deuteration can improve azobenzene performance in terms of light sensitivity, photoswitch efficiency, and photoswitch kinetics with minimal alteration to the underlying structure of the photopharmacological ligand. We report synthesized deuterated azobenzene-based ligands for the optimized optical control of ion channel and G protein-coupled receptor function in live cells, setting the stage for the straightforward, widespread adoption of this approach.

biochemistry↗

bioRxiv2023

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 can regulate 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 then identify key conserved residues and cancer-associated mutations that modulate CTD-membrane binding. Finally, we provide evidence that mGluR3 transducer coupling is controlled by CTD-membrane interactions in live cells, which may be subject to regulation by CTD phosphorylation and changes in membrane composition. 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. Significance StatementG protein-coupled receptors (GPCRs) allow cells to sense and respond to their environment and constitute the largest class of targets for approved therapeutic drugs. Temporally precise GPCR signaling is achieved by coupling the binding of extracellular ligands to the binding of intracellular signal transducers (e.g. heterotrimeric G proteins) and regulators (e.g. {beta}-arrestins). The C-terminal domains (CTDs) of GPCRs are targets of various post-translational modifications and play a critical role in transducer and regulator recruitment. Here we report novel interactions of the CTDs of two GPCRs of the metabotropic glutamate receptor family with cellular membranes. These interactions serve to regulate CTD accessibility and thus, mGluR coupling to transducers and regulators. We propose that dynamic CTD-membrane interaction constitutes a general mechanism for regulating GPCR function.

biophysics↗

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

State-dependent photocrosslinking unveiled the role of intersubunit interfaces on Ca2+ activating mechanisms of the BK channel

BK channels are high-conductance potassium channels that are activated by voltage and Ca2+. The pore-forming -subunits form homotetramers including a membrane-spanning domain and a cytosolic domain with a tandem of RCK-like domains (RCK1 and RCK2) per subunit. The eight RCKs compose high affinity Ca2+-binding sites that drive channel activation. Full-length Cryo-EM structures revealed intersubunit interactions between the RCK domains. Asparagine 449 (N449, human BK channel) is located at the RCK1 domain and coordinates Ca2+ in the RCK2 of the adjacent subunit. In addition, two Arginines (R786 and R790) of one RCK2 and Glutamate 955 of the adjacent subunit constitute an additional interaction interface. Functional studies on these residues showed that these two interfaces are crucial in Ca2+ sensitivity. To detect structural rearrangements induced by Ca2+ during channel activation, we took advantage of the photoactivatable unnatural amino acid p-benzoyl-L-phenylalanine (BzF). Functional channels were obtained with this amino acid inserted at 11 positions. N449 and R786 positions (N449BzF and R786BzF respectively). UV-induced photocrosslinking led to Ca2+ dependent and voltage-independent effects in both mutants. N449BzF showed a steady-state current reduction at saturating Ca2+ concentrations. Our data shows that this effect mainly relies on full occupancy of the RCK1 Ca2+ binding site, since mutation of this site abolished the effect. The R786BzF construct showed a substantial potentiation of the current in the absence of Ca2+. In this case, photocrosslinking seems to favor the activation of the channel by voltage. Overall, these results suggest mobile interfaces between RCK domains are key to BK channel activation.

biophysics↗

bioRxiv2020

Synaptic plasticity of somatosensory neurons is determined by functional coupling between NMDA receptors and BK channels

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 novel 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.

neuroscience↗