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G protein coupled receptors (GPCRs) exhibit varying degrees of selectivity for different G protein isoforms. Despite the abundant structures of GPCR-G protein complexes, little is known about the mechanism of G protein coupling specificity. The β2-adrenergic receptor is an example of GPCR with high selectivity for Gαs, the stimulatory G protein for adenylyl cyclase, and much weaker for the Gαi family of G proteins inhibiting adenylyl cyclase. By developing a new Gαi-biased agonist (LM189), we provide structural and biophysical evidence supporting that distinct conformations at ICL2 and TM6 are required for coupling of the different G protein subtypes Gαs and Gαi. These results deepen our understanding of G protein specificity and bias and can accelerate the design of ligands that select for preferred signaling pathways.

G protein-coupled receptors (GPCRs) within the same subfamily often share high homology in their orthosteric pocket and therefore pose challenges to drug development. The amino acids that form the orthosteric binding pocket for epinephrine and norepinephrine in the β 1 and β 2 adrenergic receptors (β 1 AR and β 2 AR) are identical. Here, to examine the effect of conformational restriction on ligand binding kinetics, we synthesized a constrained form of epinephrine. Surprisingly, the constrained epinephrine exhibits over 100-fold selectivity for the β 2 AR over the β 1 AR. We provide evidence that the selectivity may be due to reduced ligand flexibility that enhances the association rate for the β 2 AR, as well as a less stable binding pocket for constrained epinephrine in the β 1 AR. The differences in the amino acid sequence of the extracellular vestibule of the β 1 AR allosterically alter the shape and stability of the binding pocket, resulting in a marked difference in affinity compared to the β 2 AR. These studies suggest that for receptors containing identical binding pocket residues, the binding selectivity may be influenced in an allosteric manner by surrounding residues, like those of the extracellular loops (ECLs) that form the vestibule. Exploiting these allosteric influences may facilitate the development of more subtype-selective ligands for GPCRs., (© 2023. The Author(s).)

Heterotrimeric G proteins serve as membrane-associated signaling hubs, in concert with their cognate G-protein-coupled receptors. Fluorine nuclear magnetic resonance spectroscopy was employed to monitor the conformational equilibria of the human stimulatory G-protein α subunit (G s α) alone, in the intact G s αβ 1 γ 2 heterotrimer or in complex with membrane-embedded human adenosine A 2A receptor (A 2A R). The results reveal a concerted equilibrium that is strongly affected by nucleotide and interactions with the βγ subunit, the lipid bilayer and A 2A R. The α1 helix of G s α exhibits significant intermediate timescale dynamics. The α4β6 loop and α5 helix undergo membrane/receptor interactions and order-disorder transitions respectively, associated with G-protein activation. The αN helix adopts a key functional state that serves as an allosteric conduit between the βγ subunit and receptor, while a significant fraction of the ensemble remains tethered to the membrane and receptor upon activation., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

G-protein-coupled receptors (GPCRs), the largest family of signalling receptors, as well as important drug targets, are known to activate extracellular-signal-regulated kinase (ERK)-a master regulator of cell proliferation and survival 1 . However, the precise mechanisms that underlie GPCR-mediated ERK activation are not clearly understood 2-4 . Here we investigated how spatially organized β 2 -adrenergic receptor (β 2 AR) signalling controls ERK. Using subcellularly targeted ERK activity biosensors 5 , we show that β 2 AR signalling induces ERK activity at endosomes, but not at the plasma membrane. This pool of ERK activity depends on active, endosome-localized Gα s and requires ligand-stimulated β 2 AR endocytosis. We further identify an endosomally localized non-canonical signalling axis comprising Gα s , RAF and mitogen-activated protein kinase kinase, resulting in endosomal ERK activity that propagates into the nucleus. Selective inhibition of endosomal β 2 AR and Gα s signalling blunted nuclear ERK activity, MYC gene expression and cell proliferation. These results reveal a non-canonical mechanism for the spatial regulation of ERK through GPCR signalling and identify a functionally important endosomal signalling axis., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Mutations in splicing factors (SF) are the predominant class of mutations in myelodysplastic syndrome (MDS), but convergent downstream disease drivers remain elusive. To identify common direct targets of missplicing by mutant U2AF1 and SRSF2, we performed RNA sequencing and enhanced version of the cross-linking and immunoprecipitation assay in human hematopoietic stem/progenitor cells derived from isogenic induced pluripotent stem cell (iPSC) models. Integrative analyses of alternative splicing and differential binding converged on a long isoform of GNAS (GNAS-L), promoted by both mutant factors. MDS population genetics, functional and biochemical analyses support that GNAS-L is a driver of MDS and encodes a hyperactive long form of the stimulatory G protein alpha subunit, Gαs-L, that activates ERK/MAPK signaling. SF-mutant MDS cells have activated ERK signaling and consequently are sensitive to MEK inhibitors. Our findings highlight an unexpected and unifying mechanism by which SRSF2 and U2AF1 mutations drive oncogenesis with potential therapeutic implications for MDS and other SF-mutant neoplasms., Significance: SF mutations are disease-defining in MDS, but their critical effectors remain unknown. We discover the first direct target of convergent missplicing by mutant U2AF1 and SRSF2, a long GNAS isoform, which activates G protein and ERK/MAPK signaling, thereby driving MDS and rendering mutant cells sensitive to MEK inhibition. This article is highlighted in the In This Issue feature, p. 587., (©2021 American Association for Cancer Research.)

Over the past five decades, tremendous effort has been devoted to computational methods for predicting properties of ligands-i.e., molecules that bind macromolecular targets. Such methods, which are critical to rational drug design, fall into two categories: physics-based methods, which directly model ligand interactions with the target given the target's three-dimensional (3D) structure, and ligand-based methods, which predict ligand properties given experimental measurements for similar ligands. Here, we present a rigorous statistical framework to combine these two sources of information. We develop a method to predict a ligand's pose-the 3D structure of the ligand bound to its target-that leverages a widely available source of information: a list of other ligands that are known to bind the same target but for which no 3D structure is available. This combination of physics-based and ligand-based modeling improves pose prediction accuracy across all major families of drug targets. Using the same framework, we develop a method for virtual screening of drug candidates, which outperforms standard physics-based and ligand-based virtual screening methods. Our results suggest broad opportunities to improve prediction of various ligand properties by combining diverse sources of information through customized machine-learning approaches., Competing Interests: Competing interest statement: Stanford University has filed a patent application related to this work., (Copyright © 2021 the Author(s). Published by PNAS.)

Optical manipulations of genetically defined cell types have generated significant insights into the dynamics of neural circuits. While optogenetic activation has been relatively straightforward, rapid and reversible synaptic inhibition has proven more elusive. Here, we leveraged the natural ability of inhibitory presynaptic GPCRs to suppress synaptic transmission and characterize parapinopsin (PPO) as a GPCR-based opsin for terminal inhibition. PPO is a photoswitchable opsin that couples to G i/o signaling cascades and is rapidly activated by pulsed blue light, switched off with amber light, and effective for repeated, prolonged, and reversible inhibition. PPO rapidly and reversibly inhibits glutamate, GABA, and dopamine release at presynaptic terminals. Furthermore, PPO alters reward behaviors in a time-locked and reversible manner in vivo. These results demonstrate that PPO fills a significant gap in the neuroscience toolkit for rapid and reversible synaptic inhibition and has broad utility for spatiotemporal control of inhibitory GPCR signaling cascades., Competing Interests: Declaration of interests R.W.G and M.R.B. are cofounders of Neurolux Inc., a neuroscience technology company, but no related technology was used in this study. All other authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Beta adrenergic receptors (βARs) mediate physiologic responses to the catecholamines epinephrine and norepinephrine released by the sympathetic nervous system. While the hormone epinephrine binds β 1 AR and β 2 AR with similar affinity, the smaller neurotransmitter norepinephrine is approximately tenfold selective for the β 1 AR. To understand the structural basis for this physiologically important selectivity, we solved the crystal structures of the human β 1 AR bound to an antagonist carazolol and different agonists including norepinephrine, epinephrine and BI-167107. Structural comparison revealed that the catecholamine-binding pockets are identical between β 1 AR and β 2 AR, but the extracellular vestibules have different shapes and electrostatic properties. Metadynamics simulations and mutagenesis studies revealed that these differences influence the path norepinephrine takes to the orthosteric pocket and contribute to the different association rates and thus different affinities.

G-protein-coupled receptors (GPCRs) represent a ubiquitous membrane protein family and are important drug targets. Their diverse signaling pathways are driven by complex pharmacology arising from a conformational ensemble rarely captured by structural methods. Here, fluorine nuclear magnetic resonance spectroscopy ( 19 F NMR) is used to delineate key functional states of the adenosine A 2A receptor (A 2A R) complexed with heterotrimeric G protein (Gα s β 1 γ 2 ) in a phospholipid membrane milieu. Analysis of A 2A R spectra as a function of ligand, G protein, and nucleotide identifies an ensemble represented by inactive states, a G-protein-bound activation intermediate, and distinct nucleotide-free states associated with either partial- or full-agonist-driven activation. The Gβγ subunit is found to be critical in facilitating ligand-dependent allosteric transmission, as shown by 19 F NMR, biochemical, and computational studies. The results provide a mechanistic basis for understanding basal signaling, efficacy, precoupling, and allostery in GPCRs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The D2 dopamine receptor (DRD2) is a therapeutic target for Parkinson's disease 1 and antipsychotic drugs 2 . DRD2 is activated by the endogenous neurotransmitter dopamine and synthetic agonist drugs such as bromocriptine 3 , leading to stimulation of G i and inhibition of adenylyl cyclase. Here we used cryo-electron microscopy to elucidate the structure of an agonist-bound activated DRD2-G i complex reconstituted into a phospholipid membrane. The extracellular ligand-binding site of DRD2 is remodelled in response to agonist binding, with conformational changes in extracellular loop 2, transmembrane domain 5 (TM5), TM6 and TM7, propagating to opening of the intracellular G i -binding site. The DRD2-G i structure represents, to our knowledge, the first experimental model of a G-protein-coupled receptor-G-protein complex embedded in a phospholipid bilayer, which serves as a benchmark to validate the interactions seen in previous detergent-bound structures. The structure also reveals interactions that are unique to the membrane-embedded complex, including helix 8 burial in the inner leaflet, ordered lysine and arginine side chains in the membrane interfacial regions, and lipid anchoring of the G protein in the membrane. Our model of the activated DRD2 will help to inform the design of subtype-selective DRD2 ligands for multiple human central nervous system disorders.

Muscarinic toxins (MTs) are natural toxins produced by mamba snakes that primarily bind to muscarinic acetylcholine receptors (MAChRs) and modulate their function. Despite their similar primary and tertiary structures, MTs show distinct binding selectivity toward different MAChRs. The molecular details of how MTs distinguish MAChRs are not well understood. Here, we present the crystal structure of M 1 AChR in complex with MT7, a subtype-selective anti-M 1 AChR snake venom toxin. The structure reveals the molecular basis of the extreme subtype specificity of MT7 for M 1 AChR and the mechanism by which it regulates receptor function. Through in vitro engineering of MT7 finger regions that was guided by the structure, we have converted the selectivity from M 1 AChR toward M 2 AChR, suggesting that the three-finger fold is a promising scaffold for developing G protein-coupled receptor modulators., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Most drugs acting on G-protein-coupled receptors target the orthosteric binding pocket where the native hormone or neurotransmitter binds. There is much interest in finding allosteric ligands for these targets because they modulate physiologic signaling and promise to be more selective than orthosteric ligands. Here we describe a newly developed allosteric modulator of the β 2 -adrenergic receptor (β 2 AR), AS408, that binds to the membrane-facing surface of transmembrane segments 3 and 5, as revealed by X-ray crystallography. AS408 disrupts a water-mediated polar network involving E122 3.41 and the backbone carbonyls of V206 5.45 and S207 5.46 . The AS408 binding site is adjacent to a previously identified molecular switch for β 2 AR activation formed by I 3.40 , P 5.50 and F 6.44 . The structure reveals how AS408 stabilizes the inactive conformation of this switch, thereby acting as a negative allosteric modulator for agonists and positive allosteric modulator for inverse agonists.

In the version of this paper originally published, the structure for epinephrine shown in Figure 1a was redrawn with an extra carbon. The structure has been replaced in the HTML and PDF versions of the article. The original and corrected versions of the structure are shown below.

Drugs that treat chronic obstructive pulmonary disease by antagonizing the M3 muscarinic acetylcholine receptor (M3R) have had a significant effect on health, but can suffer from their lack of selectivity against the M2R subtype, which modulates heart rate. Beginning with the crystal structures of M2R and M3R, we exploited a single amino acid difference in their orthosteric binding pockets using molecular docking and structure-based design. The resulting M3R antagonists had up to 100-fold selectivity over M2R in affinity and over 1,000-fold selectivity in vivo. The crystal structure of the M3R-selective antagonist in complex with M3R corresponded closely to the docking-predicted geometry, providing a template for further optimization., Competing Interests: Conflict of interest statement: B.K.K. is a cofounder of and consultant for ConfometRx, Inc., (Copyright © 2018 the Author(s). Published by PNAS.)

The Frizzled (FZD) proteins belong to class F of G protein-coupled receptors (GPCRs) and are essential for various pathways involving the secreted lipoglycoproteins of the wingless/int-1 (WNT) family. A WNT-binding cysteine-rich domain (CRD) in FZDs is N-terminally located and connected to the seven transmembrane domain-spanning receptor core by a linker domain that has a variable length in different FZD homologs. However, the function and importance of this linker domain are poorly understood. Here we used systematic mutagenesis of FZD 6 to define the minimal N-terminal domain sufficient for receptor surface expression and recruitment of the intracellular scaffold protein Dishevelled (DVL). Further, we identified a triad of evolutionarily conserved cysteines in the FZD linker domain that is crucial for receptor membrane expression and recruitment of DVL. Our results are in agreement with the concept that the conserved cysteines in the linker domain of FZDs assist with the formation of a common secondary structure in this region. We propose that this structure could be involved in agonist binding and receptor activation mechanisms that are similar to the binding and activation mechanisms known for other GPCRs., (© 2018 Valnohova et al.)

Salmeterol is a partial agonist for the β 2 adrenergic receptor (β 2 AR) and the first long-acting β 2 AR agonist to be widely used clinically for the treatment of asthma and chronic obstructive pulmonary disease. Salmeterol's safety and mechanism of action have both been controversial. To understand its unusual pharmacological action and partial agonism, we obtained the crystal structure of salmeterol-bound β 2 AR in complex with an active-state-stabilizing nanobody. The structure reveals the location of the salmeterol exosite, where sequence differences between β 1 AR and β 2 AR explain the high receptor-subtype selectivity. A structural comparison with the β 2 AR bound to the full agonist epinephrine reveals differences in the hydrogen-bond network involving residues Ser204 5.43 and Asn293 6.55 . Mutagenesis and biophysical studies suggested that these interactions lead to a distinct active-state conformation that is responsible for the partial efficacy of G-protein activation and the limited β-arrestin recruitment for salmeterol.

G protein-coupled receptors (GPCRs) are a key drug target class. They account for over one-third of current pharmaceuticals, and both drugs that inhibit and promote receptor function are important therapeutically; in some cases, the same GPCR can be targeted with agonists and inhibitors, depending upon disease context. There have been major breakthroughs in understanding GPCR structure and drug binding through advances in X-ray crystallography, and membrane protein stabilization. Nonetheless, these structures have predominately been of inactive receptors bound to inhibitors. Efforts to capture structures of fully active GPCRs, in particular those in complex with the canonical, physiological transducer G protein, have been limited via this approach. Very recently, advances in cryo-electron microscopy have provided access to agonist:GPCR:G protein complex structures. These promise to revolutionize our understanding of GPCR:G protein engagement and provide insight into mechanisms of efficacy and coupling selectivity and how these might be controlled by biased agonists. Here we review what we have currently learned from the new GPCR:Gs and GPCR:Gi/o complex structures., Competing Interests: The authors declare no competing financial interest., (Copyright © 2018 American Chemical Society.)

The intrinsic efficacy of orthosteric ligands acting at G-protein-coupled receptors (GPCRs) reflects their ability to stabilize active receptor states (R*) and is a major determinant of their physiological effects. Here, we present a direct way to quantify the efficacy of ligands by measuring the binding of a R*-specific biosensor to purified receptor employing interferometry. As an example, we use the mu-opioid receptor (µ-OR), a prototypic class A GPCR, and its active state sensor, nanobody-39 (Nb39). We demonstrate that ligands vary in their ability to recruit Nb39 to µ-OR and describe methadone, loperamide, and PZM21 as ligands that support unique R* conformation(s) of µ-OR. We further show that positive allosteric modulators of µ-OR promote formation of R* in addition to enhancing promotion by orthosteric agonists. Finally, we demonstrate that the technique can be utilized with heterotrimeric G protein. The method is cell-free, signal transduction-independent and is generally applicable to GPCRs., Competing Interests: KL, JM, AM, RS, JT No competing interests declared, (© 2018, Livingston et al.)

Cations play key roles in regulating G-protein-coupled receptors (GPCRs), although their mechanisms are poorly understood. Here, 19 F NMR is used to delineate the effects of cations on functional states of the adenosine A 2A GPCR. While Na + reinforces an inactive ensemble and a partial-agonist stabilized state, Ca 2+ and Mg 2+ shift the equilibrium toward active states. Positive allosteric effects of divalent cations are more pronounced with agonist and a G-protein-derived peptide. In cell membranes, divalent cations enhance both the affinity and fraction of the high affinity agonist-bound state. Molecular dynamics simulations suggest high concentrations of divalent cations bridge specific extracellular acidic residues, bringing TM5 and TM6 together at the extracellular surface and allosterically driving open the G-protein-binding cleft as shown by rigidity-transmission allostery theory. An understanding of cation allostery should enable the design of allosteric agents and enhance our understanding of GPCR regulation in the cellular milieu.

Subtype-selective antagonists for muscarinic acetylcholine receptors (mAChRs) have long been elusive, owing to the highly conserved orthosteric binding site. However, allosteric sites of these receptors are less conserved, motivating the search for allosteric ligands that modulate agonists or antagonists to confer subtype selectivity. Accordingly, a 4.6 million-molecule library was docked against the structure of the prototypical M 2 mAChR, seeking molecules that specifically stabilized antagonist binding. This led us to identify a positive allosteric modulator (PAM) that potentiated the antagonist N -methyl scopolamine (NMS). Structure-based optimization led to compound '628, which enhanced binding of NMS, and the drug scopolamine itself, with a cooperativity factor (α) of 5.5 and a K B of 1.1 μM, while sparing the endogenous agonist acetylcholine. NMR spectral changes determined for methionine residues reflected changes in the allosteric network. Moreover, '628 slowed the dissociation rate of NMS from the M 2 mAChR by 50-fold, an effect not observed at the other four mAChR subtypes. The specific PAM effect of '628 on NMS antagonism was conserved in functional assays, including agonist stimulation of [ 35 S]GTPγS binding and ERK 1/2 phosphorylation. Importantly, the selective allostery between '628 and NMS was retained in membranes from adult rat hypothalamus and in neonatal rat cardiomyocytes, supporting the physiological relevance of this PAM/antagonist approach. This study supports the feasibility of discovering PAMs that confer subtype selectivity to antagonists; molecules like '628 can convert an armamentarium of potent but nonselective GPCR antagonist drugs into subtype-selective reagents, thus reducing their off-target effects., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Muscarinic receptor agonists are characterized by apparently strict restraints on their tertiary or quaternary amine and their distance to an ester or related center. On the basis of the active state crystal structure of the muscarinic M 2 receptor in complex with iperoxo, we explored potential agonists that lacked the highly conserved functionalities of previously known ligands. Using structure-guided pharmacophore design followed by docking, we found two agonists (compounds 3 and 17), out of 19 docked and synthesized compounds, that fit the receptor well and were predicted to form a hydrogen-bond conserved among known agonists. Structural optimization led to compound 28, which was 4-fold more potent than its parent 3. Fortified by the discovery of this new scaffold, we sought a broader range of chemotypes by docking 2.2 million fragments, which revealed another three micromolar agonists unrelated either to 28 or known muscarinics. Even pockets as tightly defined and as deeply studied as that of the muscarinic reveal opportunities for the structure-based design and the discovery of new chemotypes.

FRET and BRET approaches are well established for detecting ligand induced GPCR-G protein interactions in cells. Currently, FRET/BRET assays rely on co-expression of GPCR and G protein, and hence depend on the stoichiometry and expression levels of the donor and acceptor probes. On the other hand, GPCR-G protein fusions have been used extensively to understand the selectivity of GPCR signaling pathways. However, the signaling properties of fusion proteins are not consistent across GPCRs. In this study, we describe and characterize novel sensors based on the Systematic Protein Affinity Strength Modulation (SPASM) technique. Sensors consist of a GPCR and G protein tethered by an ER/K linker flanked by FRET probes. SPASM sensors are tested for the β2-, α1-, and α2- adrenergic receptors, and adenosine type 1 receptor (A 1 R), tethered to Gαs-XL, Gαi 2 , or Gαq subunits. Agonist stimulation of β2-AR and α2-AR increases FRET signal comparable to co-expressed FRET/BRET sensors. SPASM sensors also retain signaling through the endogenous G protein milieu. Importantly, ER/K linker length systematically tunes the GPCR-G protein interaction, with consequent modulation of second messenger signaling for cognate interactions. SPASM GPCR sensors serve the dual purpose of detecting agonist-induced changes in GPCR-G protein interactions, and linking these changes to downstream signaling.

Family B G protein-coupled receptors (GPCRs) play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1) receptor (GLP1R), whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL) particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms.

Cell transmembrane receptors play a key role in the detection of environmental stimuli and control of intracellular communication. G protein-coupled receptors constitute the largest transmembrane protein family involved in cell signaling. However, current methods for their functional reconstitution in biomimetic membranes remain both challenging and limited in scope. Herein, we describe the spontaneous reconstitution of adenosine A 2A receptor (A 2A R) during the de novo formation of synthetic liposomes via native chemical ligation. The approach takes advantage of a nonenzymatic and chemoselective method to rapidly generate A 2A R embedded phospholiposomes from receptor solubilized in n-dodecyl-β-d-maltoside analogs. In situ lipid synthesis for protein reconstitution technology proceeds in the absence of dialysis and/or detergent absorbents, and A 2A R assimilation into synthetic liposomes can be visualized by microscopy and probed by radio-ligand binding.

G protein-coupled receptors (GPCRs) respond to extracellular stimuli and interact with several intracellular binding partners to elicit cellular responses, including heterotrimeric G proteins. Recent structural and biophysical studies have highlighted the dynamic nature of GPCRs and G proteins and have identified specific conformational changes important for receptor-mediated nucleotide exchange on Gα. While domain separation within Gα is necessary for GDP release, opening the inter-domain interface is insufficient to stimulate nucleotide exchange. Rather, an activated receptor promotes GDP release by allosterically disrupting the nucleotide-binding site via interactions with the Gα N-termini and C-termini. Highlighting the allosteric nature of GPCRs, recent studies suggest that agonist binding alone poorly stabilizes an active conformation of several receptors. Rather, full stabilization of the receptor in an active state requires formation of the agonist-receptor-G protein ternary complex. In turn, nucleotide-free Gα is able to stabilize conformational changes around the receptor's agonist-binding site to enhance agonist affinity., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Classically, G protein-coupled receptor (GPCR) stimulation promotes G protein signaling at the plasma membrane, followed by rapid β-arrestin-mediated desensitization and receptor internalization into endosomes. However, it has been demonstrated that some GPCRs activate G proteins from within internalized cellular compartments, resulting in sustained signaling. We have used a variety of biochemical, biophysical, and cell-based methods to demonstrate the existence, functionality, and architecture of internalized receptor complexes composed of a single GPCR, β-arrestin, and G protein. These super-complexes or "megaplexes" more readily form at receptors that interact strongly with β-arrestins via a C-terminal tail containing clusters of serine/threonine phosphorylation sites. Single-particle electron microscopy analysis of negative-stained purified megaplexes reveals that a single receptor simultaneously binds through its core region with G protein and through its phosphorylated C-terminal tail with β-arrestin. The formation of such megaplexes provides a potential physical basis for the newly appreciated sustained G protein signaling from internalized GPCRs., (Copyright © 2016 Elsevier Inc. All rights reserved.)

G-protein-coupled receptors (GPCRs) remain the primary conduit by which cells detect environmental stimuli and communicate with each other. Upon activation by extracellular agonists, these seven-transmembrane-domain-containing receptors interact with heterotrimeric G proteins to regulate downstream second messenger and/or protein kinase cascades. Crystallographic evidence from a prototypic GPCR, the β2-adrenergic receptor (β2AR), in complex with its cognate G protein, Gs, has provided a model for how agonist binding promotes conformational changes that propagate through the GPCR and into the nucleotide-binding pocket of the G protein α-subunit to catalyse GDP release, the key step required for GTP binding and activation of G proteins. The structure also offers hints about how G-protein binding may, in turn, allosterically influence ligand binding. Here we provide functional evidence that G-protein coupling to the β2AR stabilizes a ‘closed’ receptor conformation characterized by restricted access to and egress from the hormone-binding site. Surprisingly, the effects of G protein on the hormone-binding site can be observed in the absence of a bound agonist, where G-protein coupling driven by basal receptor activity impedes the association of agonists, partial agonists, antagonists and inverse agonists. The ability of bound ligands to dissociate from the receptor is also hindered, providing a structural explanation for the G-protein-mediated enhancement of agonist affinity, which has been observed for many GPCR–G-protein pairs. Our data also indicate that, in contrast to agonist binding alone, coupling of a G protein in the absence of an agonist stabilizes large structural changes in a GPCR. The effects of nucleotide-free G protein on ligand-binding kinetics are shared by other members of the superfamily of GPCRs, suggesting that a common mechanism may underlie G-protein-mediated enhancement of agonist affinity.

The recent, unfortunate death of Alfred G. ("Al") Gilman, M.D., Ph.D., represents a sad signpost for an era spanning over 40 years in molecular pharmacology. Gilman's discoveries, influence, and persona were dominant forces in research and training in pharmacology. Here, we review the progression of ideas and knowledge that spawned early work by Gilman and collaborators (among them, one of the authors) and later efforts (including those of the other author) that have recently yielded a comprehensive and precise structural understanding of fundamental topics in pharmacology: the binding of ligands to G protein-coupled receptors (GPCRs) and the interaction of GPCRs with heterotrimeric G proteins and effector molecules. Those data provide new and important insights into the molecular basis that underlies affinity and efficacy, two of the most important features of drug action, which represent the latest chapter in the saga that Al Gilman's work helped launch., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

DNA provides an ideal substrate for the engineering of versatile nanostructures due to its reliable Watson-Crick base pairing and well-characterized conformation. One of the most promising applications of DNA nanostructures arises from the site-directed spatial arrangement with nanometer precision of guest components such as proteins, metal nanoparticles, and small molecules. Two-dimensional DNA origami architectures, in particular, offer a simple design, high yield of assembly, and large surface area for use as a nanoplatform. However, such single-layer DNA origami were recently found to be structurally polymorphous due to their high flexibility, leading to the development of conformationally restrained multilayered origami that lack some of the advantages of the single-layer designs. Here we monitored single-layer DNA origami by transmission electron microscopy (EM) and discovered that their conformational heterogeneity is dramatically reduced in the presence of a low concentration of dimethyl sulfoxide, allowing for an efficient flattening onto the carbon support of an EM grid. We further demonstrated that streptavidin and a biotinylated target protein (cocaine esterase, CocE) can be captured at predesignated sites on these flattened origami while maintaining their functional integrity. Our demonstration that protein assemblies can be constructed with high spatial precision (within ∼2 nm of their predicted position on the platforms) by using strategically flattened single-layer origami paves the way for exploiting well-defined guest molecule assemblies for biochemistry and nanotechnology applications.

G protein-coupled receptors (GPCRs) relay diverse extracellular signals into cells by catalyzing nucleotide release from heterotrimeric G proteins, but the mechanism underlying this quintessential molecular signaling event has remained unclear. Here we use atomic-level simulations to elucidate the nucleotide-release mechanism. We find that the G protein α subunit Ras and helical domains-previously observed to separate widely upon receptor binding to expose the nucleotide-binding site-separate spontaneously and frequently even in the absence of a receptor. Domain separation is necessary but not sufficient for rapid nucleotide release. Rather, receptors catalyze nucleotide release by favoring an internal structural rearrangement of the Ras domain that weakens its nucleotide affinity. We use double electron-electron resonance spectroscopy and protein engineering to confirm predictions of our computationally determined mechanism., (Copyright © 2015, American Association for the Advancement of Science.)

Although several recent studies have reported that GPCRs adopt multiple conformations, it remains unclear how subtle conformational changes are translated into divergent downstream responses. In this study, we report on a novel class of FRET-based sensors that can detect the ligand/mutagenic stabilization of GPCR conformations that promote interactions with G proteins in live cells. These sensors rely on the well characterized interaction between a GPCR and the C terminus of a Gα subunit. We use these sensors to elucidate the influence of the highly conserved (E/D)RY motif on GPCR conformation. Specifically, Glu/Asp but not Arg mutants of the (E/D)RY motif are known to enhance basal GPCR signaling. Hence, it is unclear whether ionic interactions formed by the (E/D)RY motif (ionic lock) are necessary to stabilize basal GPCR states. We find that mutagenesis of the β2-AR (E/D)RY ionic lock enhances interaction with Gs. However, only Glu/Asp but not Arg mutants increase G protein activation. In contrast, mutagenesis of the opsin (E/D)RY ionic lock does not alter its interaction with transducin. Instead, opsin-specific ionic interactions centered on residue Lys-296 are both necessary and sufficient to promote interactions with transducin. Effective suppression of β2-AR basal activity by inverse agonist ICI 118,551 requires ionic interactions formed by the (E/D)RY motif. In contrast, the inverse agonist metoprolol suppresses interactions with Gs and promotes Gi binding, with concomitant pertussis toxin-sensitive inhibition of adenylyl cyclase activity. Taken together, these studies validate the use of the new FRET sensors while revealing distinct structural mechanisms for ligand-dependent GPCR function.

A long-held tenet of molecular pharmacology is that canonical signal transduction mediated by G-protein-coupled receptor (GPCR) coupling to heterotrimeric G proteins is confined to the plasma membrane. Evidence supporting this traditional view is based on analytical methods that provide limited or no subcellular resolution. It has been subsequently proposed that signalling by internalized GPCRs is restricted to G-protein-independent mechanisms such as scaffolding by arrestins, or GPCR activation elicits a discrete form of persistent G protein signalling, or that internalized GPCRs can indeed contribute to the acute G-protein-mediated response. Evidence supporting these various latter hypotheses is indirect or subject to alternative interpretation, and it remains unknown if endosome-localized GPCRs are even present in an active form. Here we describe the application of conformation-specific single-domain antibodies (nanobodies) to directly probe activation of the β2-adrenoceptor, a prototypical GPCR, and its cognate G protein, Gs (ref. 12), in living mammalian cells. We show that the adrenergic agonist isoprenaline promotes receptor and G protein activation in the plasma membrane as expected, but also in the early endosome membrane, and that internalized receptors contribute to the overall cellular cyclic AMP response within several minutes after agonist application. These findings provide direct support for the hypothesis that canonical GPCR signalling occurs from endosomes as well as the plasma membrane, and suggest a versatile strategy for probing dynamic conformational change in vivo.

GPCRs mediate intracellular signaling upon external stimuli, making them ideal drug targets. However, little is known about their activation mechanisms due to the difficulty in purification. Here, we introduce a method to purify GPCRs in nanodiscs, which incorporates GPCRs into lipid bilayers immediately after membrane solubilization, followed by single-step purification. Using this approach, we purified a family B GPCR, parathyroid hormone 1 receptor (PTH1R), which regulates calcium and phosphate homeostasis and is a drug target for osteoporosis. We demonstrated that the purified PTH1R in nanodiscs can bind to PTH(1-34) and activate G protein. We also observed that Ca(2+) is a weak agonist of PTH1R, and Ca(2+) in millimolar concentration can switch PTH(1-34) from an inverse agonist to an agonist. Hence, our results show that nanodiscs are a viable vehicle for GPCR purification, enabling studies of GPCRs under precise experimental conditions without interference from other cellular or membrane components.

G protein-coupled receptors (GPCRs) have critical roles in various physiological and pathophysiological processes, and more than 40% of marketed drugs target GPCRs. Although the canonical downstream target of an agonist-activated GPCR is a G protein heterotrimer; there is a growing body of evidence suggesting that other signaling molecules interact, directly or indirectly, with GPCRs. However, due to the low abundance in the intact cell system and poor solubility of GPCRs, identification of these GPCR-interacting molecules remains challenging. Here, we establish a strategy to overcome these difficulties by using high-density lipoprotein (HDL) particles. We used the β(2)-adrenergic receptor (β(2)AR), a GPCR involved in regulating cardiovascular physiology, as a model system. We reconstituted purified β(2)AR in HDL particles, to mimic the plasma membrane environment, and used the reconstituted receptor as bait to pull-down binding partners from rat heart cytosol. A total of 293 proteins were identified in the full agonist-activated β(2)AR pull-down, 242 proteins in the inverse agonist-activated β(2)AR pull-down, and 210 proteins were commonly identified in both pull-downs. A small subset of the β(2)AR-interacting proteins isolated was confirmed by Western blot; three known β(2)AR-interacting proteins (Gsα, NHERF-2, and Grb2) and 3 newly identified known β(2)AR-interacting proteins (AMPKα, acetyl-CoA carboxylase, and UBC-13). Profiling of the identified proteins showed a clear bias toward intracellular signal transduction pathways, which is consistent with the role of β(2)AR as a cell signaling molecule. This study suggests that HDL particle-reconstituted GPCRs can provide an effective platform method for the identification of GPCR binding partners coupled with a mass spectrometry-based proteomic analysis.

Previous studies have demonstrated the capacity of a long-acting mutant form of a naturally occurring bacterial double mutant cocaine esterase (DM CocE) to antagonize the reinforcing, discriminative, convulsant, and lethal effects of cocaine in rodents and reverse the increases in mean arterial pressure (MAP) and heart rate (HR) produced by cocaine in rhesus monkeys. This study was aimed at characterizing the immunologic responses to repeated dosing with DM CocE and determining whether the development of anti-CocE antibodies altered the capacity of DM CocE to reduce plasma cocaine levels and ameliorate the cardiovascular effects of cocaine in rhesus monkeys. Under control conditions, intravenous administration of cocaine (3 mg/kg) resulted in a rapid increase in the plasma concentration of cocaine (n = 2) and long-lasting increases in MAP and HR (n = 3). Administration of DM CocE (0.32 mg/kg i.v.) 10 min after cocaine resulted in a rapid hydrolysis of cocaine with plasma levels below detection limits within 5 to 8 min. Elevations in MAP and HR were significantly reduced within 25 and 50 min of DM CocE administration, respectively. Although slight (10-fold) increases in anti-CocE antibodies were observed after the fourth administration of DM CocE, these antibodies did not alter the capacity of DM CocE to reduce plasma cocaine levels or ameliorate cocaine's cardiovascular effects. Anti-CocE titers were transient and generally dissipated within 8 weeks. Together, these results suggest that highly efficient cocaine esterases, such as DM CocE, may provide a novel and effective therapeutic for the treatment of acute cocaine intoxication in humans.

Cocaine esterase (CocE) is the most efficient cocaine-metabolizing enzyme tested in vivo to date, displaying a rapid clearance of cocaine and a robust protection against cocaine's toxicity. Two potential obstacles to the clinical application of CocE, however, lie in its proteolytic degradation and induced immune response. To minimize these potential obstacles, we attempted nondisruptive cell encapsulation by creating a cell permeable form of CocE, which was achieved by covalently linking a thermally stable CocE mutant (dmCocE) with cell penetrating peptides (CPPs). Two types of CPPs, Tat and the low molecular weight protamine (LMWP), were used in this study. Two types of disulfide-bridged chemical conjugates, Tat-S-S-dmCocE and LMWP-S-S-dmCocE, were synthesized and then purified by heparin affinity chromatography. In addition, four recombinant CPP-dmCocE fusion proteins, Tat-N-dmCocE, LMWP-N-dmCocE, dmCocE-C-Tat, and dmCocE-C-LMWP, were constructed, expressed in Escherichia coli, and purified as soluble proteins. Among these six CPP-dmCocE variants, LMWP-S-S-dmCocE showed the highest cocaine-hydrolyzing activity, and dmCocE-C-Tat had the highest production yield. To evaluate their cellular uptake behavior, a covalently linked fluorophore (FITC) was utilized to visualize the cellular uptake of all six CPP-dmCocE variants in living HeLa cells. All the six variants exhibited cellular uptake, but their intracellular distribution phenotypes differed. While the chemical conjugates showed primarily cytoplasmic distribution, which was likely due to the reduction of the disulfide linkage between CPP and dmCocE, all the other four recombinant fusion proteins displayed both nuclear and cytoplasmic localization, with dmCocE-C-CPP exhibiting higher cytoplasmic distribution during cellular uptake. Based on a balanced consideration of essentials for clinical application, including parameters such as high cocaine-hydrolyzing efficiency, large production yield, major cytoplasmic distribution, etc., the dmCocE-C-Tat fusion protein seems to be the best candidate from this investigation. Further in vivo studies of the cell-encapsulated dmCocE-C-Tat in hydrolyzing cocaine and alleviating immunogenicity and proteolytic degradation in established, clinically relevant mouse models are currently underway in our laboratories. Findings from this research are not only useful for developing other new CPP-CocE constructs but also valuable for establishing a nondisruptive cell-encapsulation technology for other protein therapeutics that are known to be immunogenic for direct clinical application.

Recent mutagenesis studies have identified a mutant G4C/S10C/T172R/G173Q cocaine esterase (CCRQ CocE) with an in vitro duration of action of >40 days. Although the in vivo duration of CCRQ CocE's action was <24 h, modification of this enzyme with polyethylene glycol (PEG) polymers resulted in a CocE (PEG-CCRQ CocE) capable of preventing cocaine-induced lethality for up to 72 h. The current studies were aimed at providing a detailed characterization of the effectiveness, selectivity, and duration of PEG-CCRQ CocE's actions in cocaine self-administration and discrimination assays in rats. Pretreatment with PEG-CCRQ CocE produced dose-dependent rightward shifts in the dose-response curves for cocaine self-administration and discrimination, with the highest dose of PEG-CCRQ CocE capable of producing an initial shift of cocaine's reinforcing and interoceptive effects of >30-fold to the right, with significant inhibition of these effects observed for up to 72 h. Although PEG-CCRQ CocE also produced slight reductions in the rates of methylphenidate- and food-reinforced responding, these effects were short-lived, lasting <24 h. Finally, when taken together with the finding that PEG-CCRQ CocE failed to alter the cocaine-like interoceptive effects of either methylphenidate or d-amphetamine, these results suggest that PEG-CCRQ CocE possesses a high degree of pharmacologic specificity for cocaine and a prolonged in vivo duration of action. In conclusion, these studies provide strong evidence to support the further development of long-lasting, highly efficient CocEs, such as PEG-CCRQ CocE, as a potential therapeutic option for the treatment of cocaine abuse in humans.

Opium is one of the world's oldest drugs, and its derivatives morphine and codeine are among the most used clinical drugs to relieve severe pain. These prototypical opioids produce analgesia as well as many undesirable side effects (sedation, apnoea and dependence) by binding to and activating the G-protein-coupled µ-opioid receptor (µ-OR) in the central nervous system. Here we describe the 2.8 Å crystal structure of the mouse µ-OR in complex with an irreversible morphinan antagonist. Compared to the buried binding pocket observed in most G-protein-coupled receptors published so far, the morphinan ligand binds deeply within a large solvent-exposed pocket. Of particular interest, the µ-OR crystallizes as a two-fold symmetrical dimer through a four-helix bundle motif formed by transmembrane segments 5 and 6. These high-resolution insights into opioid receptor structure will enable the application of structure-based approaches to develop better drugs for the management of pain and addiction.

Cocaine is highly addictive and there are no pharmacotherapeutic drugs available to treat acute cocaine toxicity or chronic abuse. Antagonizing an inhibitor such as cocaine using a small molecule has proven difficult. The alternative approach is to modify cocaine's pharmacokinetic properties by sequestering or hydrolyzing it in serum and limiting access to its sites of action. We took advantage of a bacterial esterase (CocE) that has evolved to hydrolyze cocaine and have developed it as a therapeutic that rapidly and specifically clears cocaine from the subject. Native enzyme was unstable at 37°C, thus limiting CocE's potential. Innovative computational methods based on the protein's structure helped elucidate its mechanism of destabilization. Novel protein engineering methodologies were applied to substantially improve its stability in vitro and in vivo. These improvements rendered CocE as a powerful and efficacious therapeutic to treat cocaine intoxication and lead the way towards developing a therapy for addiction.

Cocaine abuse and toxicity remain widespread problems in the United States. Currently cocaine toxicity is treated only symptomatically, because there is no Food and Drug Administration-approved pharmacotherapy for this indication. To address the unmet need, a stabilized mutant of bacterial cocaine esterase [T172R/G173Q-CocE (DM-CocE)], which hydrolyzes cocaine into inactive metabolites and has low immunogenic potential, has been developed and previously tested in animal models of cocaine toxicity. Here, we document the rapid cocaine hydrolysis by low doses of DM-CocE in vitro and in vivo, as well as the pharmacokinetics and distribution of the DM-CocE protein in rats. DM-CocE at 50.5 μg/kg effectively eliminated 4 mg/kg cocaine within 2 min in both male and female rats as measured by mass spectrometry. We expanded on these findings by using a pharmacologically relevant dose of DM-CocE (0.32 mg/kg) in rats and monkeys to hydrolyze convulsant doses of cocaine. DM-CocE reduced cocaine to below detection limits rapidly after injection; however, elimination of DM-CocE resulted in peripheral cocaine redistribution by 30 to 60 min. Elimination of DM-CocE was quantified by using [³⁵S] labeling of the enzyme and was found to have a half-life of 2.1 h in rats. Minor urinary output of DM-CocE was also observed. Immunohistochemistry, Western blotting, and radiography all were used to elucidate the mechanism of DM-CocE elimination, rapid proteolysis, and recycling of amino acids into all tissues. This rapid elimination of DM-CocE is a desirable property of a therapeutic for cocaine toxicity and should reduce the likelihood of immunogenic or adverse reactions as DM-CocE moves toward clinical use.

Background: Cocaine toxicity is a prevalent problem in the Unites States for which there is currently no FDA-approved pharmacotherapy. We have developed a bacterial cocaine esterase (CocE) towards this indication. A thermostabilized mutant of CocE (DM-CocE) retains the hydrolytic activity of the wild-type esterase, rapidly hydrolyzing cocaine into the inactive metabolites ecgonine methyl ester and benzoic acid, and can prevent cocaine toxicities in rodent and non-human primate models. To advance DM-CocE towards clinical use, we examine here how the hydrolytic activity of DM-CocE is altered by some drugs commonly co-administered with cocaine., Methods: We employed a spectrophotometric cocaine hydrolysis assay to evaluate whether pharmacologically relevant doses of alcohol, nicotine, morphine, phencyclidine, ketamine, methamphetamine, naltrexone, naloxone, or midazolam would alter the Michaelis-Menten kinetics of DM-CocE for cocaine. Mass spectrometry was used to evaluate interaction with diazepam as this drug interferes with the absorbance spectra of cocaine critical for the spectrophotometric assay., Results: Alcohol, nicotine, morphine, phencyclidine, ketamine, methamphetamine, naltrexone, naloxone, and midazolam did not alter cocaine hydrolysis by DM-CocE. However, diazepam significantly slowed DM-CocE cocaine hydrolysis at very high concentrations, most likely through interaction of the phenyl ring of the benzodiazepine with the active site of DM-CocE., Conclusions: DM-CocE does not display significant drug interactions, with the exception of diazepam, which may warrant further study as DM-CocE progresses towards a clinically used pharmacotherapy for cocaine toxicity. Alternate benzodiazepines, e.g., midazolam could be used to avoid this potential interaction., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Cocaine toxicity is a widespread problem in the United States, responsible for more than 500,000 emergency department visits a year. There is currently no U.S. Food and Drug Administration-approved pharmacotherapy to directly treat cocaine toxicity. To this end, we have developed a mutant bacterial cocaine esterase (DM-CocE), which has been previously shown to rapidly hydrolyze cocaine into inert metabolites, preventing and reversing toxicity with limited immunogenic potential. Herein we describe the ability of DM-CocE to hydrolyze the active cocaine metabolites norcocaine and cocaethylene and its inability to hydrolyze benzoylecgonine. DM-CocE hydrolyzes norcocaine and cocaethylene with 58 and 45% of its catalytic efficiency for cocaine in vitro as measured by a spectrophotometric assay. We have developed a mass spectrometry method to simultaneously detect cocaine, benzoylecgonine, norcocaine, and ecgonine methyl ester to quantify the effect of DM-CocE on normal cocaine metabolism in vivo. DM-CocE administered to rats 10 min after a convulsant dose of cocaine alters the normal metabolism of cocaine, rapidly decreasing circulating levels of cocaine and norcocaine while increasing ecgonine methyl ester formation. Benzoylecgonine was not hydrolyzed in vivo, but circulating concentrations were reduced, suggesting that DM-CocE may bind and sequester this metabolite. These findings suggest that DM-CocE may reduce cocaine toxicity by eliminating active and toxic metabolites along with the parent cocaine molecule.

No small-molecule therapeutic is available to treat cocaine addiction, but enzyme-based therapy to accelerate cocaine hydrolysis in serum has gained momentum. Bacterial cocaine esterase (CocE) is the fastest known native enzyme that hydrolyzes cocaine. However, its lability at 37°C has limited its therapeutic potential. Cross-linking subunits through disulfide bridging is commonly used to stabilize multimeric enzymes. Herein we use structural methods to guide the introduction of two cysteine residues within dimer interface of CocE to facilitate intermolecular disulfide bond formation. The disulfide-crosslinked enzyme displays improved thermostability, particularly when combined with previously described mutations that enhance stability (T172R-G173Q). The newly modified enzyme yielded an extremely stable form of CocE (CCRQ-CocE) that retained greater than 90% of its activity after 41 days at 37°C, representing an improvement of more than 4700-fold over the wild-type enzyme. CCRQ-CocE could also be modified by polyethylene glycol (PEG) polymers, which improved its in vivo residence time from 24 to 72 h, as measured by a cocaine lethality assay, by self-administration in rodents, and by measurement of inhibition of cocaine-induced cardiovascular effects in rhesus monkeys. PEG-CCRQ elicited negligible immune response in rodents. Subunit stabilization and PEGylation has thus produced a potential protein therapeutic with markedly higher stability both in vitro and in vivo.

Background: A longer acting, double mutant bacterial cocaine esterase (CocE T172R/G173Q; DM CocE) has been shown to protect mice from cocaine-induced lethality, inhibit the reinforcing effects of cocaine in rats, and reverse cocaine's cardiovascular effects in rhesus monkeys. The current studies evaluated the effectiveness of DM CocE to protect against, and reverse cocaine's cardiovascular, convulsant, and lethal effects in male and female rats., Methods: Pretreatment studies were used to determine the effectiveness and in vivo duration of action for DM CocE to protect rats against the occurrence of cardiovascular changes, convulsion and lethality associated with acute cocaine toxicity. Posttreatment studies were used to evaluate the capacity of DM CocE to rescue rats from the cardiovascular and lethal effects of large doses of cocaine. In addition, male and female rats were studied to determine if there were any potential effects of sex on the capacity of DM CocE to protect against, or reverse acute cocaine toxicity in rats., Results: Pretreatment with DM CocE dose-dependently protected rats against cocaine-induced cardiovascular changes, convulsion and lethality, with higher doses active for up to 4h, and shifting cocaine-induced lethality at least 10-fold to the right. In addition to dose-dependently recovering rats from an otherwise lethal dose of cocaine, post-treatment with DM CocE also reversed the cardiovascular effects of cocaine. There were no sex-related differences in the effectiveness of DM CocE to protect against, or reverse acute cocaine toxicity., Conclusions: Together, these results support the development of DM CocE for the treatment of acute cocaine toxicity., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

G protein-coupled receptors represent the largest family of membrane receptors that instigate signalling through nucleotide exchange on heterotrimeric G proteins. Nucleotide exchange, or more precisely, GDP dissociation from the G protein α-subunit, is the key step towards G protein activation and initiation of downstream signalling cascades. Despite a wealth of biochemical and biophysical studies on inactive and active conformations of several heterotrimeric G proteins, the molecular underpinnings of G protein activation remain elusive. To characterize this mechanism, we applied peptide amide hydrogen-deuterium exchange mass spectrometry to probe changes in the structure of the heterotrimeric bovine G protein, Gs (the stimulatory G protein for adenylyl cyclase) on formation of a complex with agonist-bound human β(2) adrenergic receptor (β(2)AR). Here we report structural links between the receptor-binding surface and the nucleotide-binding pocket of Gs that undergo higher levels of hydrogen-deuterium exchange than would be predicted from the crystal structure of the β(2)AR-Gs complex. Together with X-ray crystallographic and electron microscopic data of the β(2)AR-Gs complex (from refs 2, 3), we provide a rationale for a mechanism of nucleotide exchange, whereby the receptor perturbs the structure of the amino-terminal region of the α-subunit of Gs and consequently alters the 'P-loop' that binds the β-phosphate in GDP. As with the Ras family of small-molecular-weight G proteins, P-loop stabilization and β-phosphate coordination are key determinants of GDP (and GTP) binding affinity., (© 2011 Macmillan Publishers Limited. All rights reserved)

The active-state complex between an agonist-bound receptor and a guanine nucleotide-free G protein represents the fundamental signaling assembly for the majority of hormone and neurotransmitter signaling. We applied single-particle electron microscopy (EM) analysis to examine the architecture of agonist-occupied β(2)-adrenoceptor (β(2)AR) in complex with the heterotrimeric G protein Gs (Gαsβγ). EM 2D averages and 3D reconstructions of the detergent-solubilized complex reveal an overall architecture that is in very good agreement with the crystal structure of the active-state ternary complex. Strikingly however, the α-helical domain of Gαs appears highly flexible in the absence of nucleotide. In contrast, the presence of the pyrophosphate mimic foscarnet (phosphonoformate), and also the presence of GDP, favor the stabilization of the α-helical domain on the Ras-like domain of Gαs. Molecular modeling of the α-helical domain in the 3D EM maps suggests that in its stabilized form it assumes a conformation reminiscent to the one observed in the crystal structure of Gαs-GTPγS. These data argue that the α-helical domain undergoes a nucleotide-dependent transition from a flexible to a conformationally stabilized state.

G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The β(2) adrenergic receptor (β(2)AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β(2)AR and nucleotide-free Gs heterotrimer. The principal interactions between the β(2)AR and Gs involve the amino- and carboxy-terminal α-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the β(2)AR include a 14 Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an α-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the α-helical domain of Gαs relative to the Ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR., (© 2011 Macmillan Publishers Limited. All rights reserved)

A long-acting mutant form of a naturally occurring bacterial cocaine esterase (T172R/G173Q CocE; double mutant CocE (DM CocE)) has previously been shown to antagonize the reinforcing, convulsant, and lethal effects of cocaine in rodents. However, the effectiveness and therapeutic characteristics of DM CocE in nonhuman primates, in a more clinically relevant context, are unknown. The current studies were aimed at (1) characterizing the cardiovascular effects of cocaine in freely moving rhesus monkeys, (2) evaluating the capacity of DM CocE to ameliorate these cocaine-induced cardiovascular effects when administered 10 min after cocaine, and (3) assessing the immunological responses of monkeys to DM CocE following repeated administration. Intravenous administration of cocaine produced dose-dependent increases in mean arterial pressure (MAP) and heart rate (HR) that persisted throughout the 2-h observation period following a dose of 3.2 mg/kg cocaine. Cocaine failed to produce reliable changes in electrocardiograph (ECG) parameters, body temperature, and locomotor activity. DM CocE produced a rapid and dose-dependent amelioration of the cardiovascular effects, with saline-like MAP measures restored within 5-10 min, and saline-like HR measures restored within 20-40 min of DM CocE administration. Although administration of DM CocE produced increases in anti-CocE antibodies, they did not appear to have a neutralizing effect on the capacity of DM CocE to reverse the cardiovascular effects of cocaine. In conclusion, these findings in monkeys provide strong evidence to suggest that highly efficient cocaine esterases, such as DM CocE, can provide a potential therapeutic option for treatment of acute cocaine intoxication in humans.

G protein coupled receptors (GPCRs) exhibit a spectrum of functional behaviours in response to natural and synthetic ligands. Recent crystal structures provide insights into inactive states of several GPCRs. Efforts to obtain an agonist-bound active-state GPCR structure have proven difficult due to the inherent instability of this state in the absence of a G protein. We generated a camelid antibody fragment (nanobody) to the human β(2) adrenergic receptor (β(2)AR) that exhibits G protein-like behaviour, and obtained an agonist-bound, active-state crystal structure of the receptor-nanobody complex. Comparison with the inactive β(2)AR structure reveals subtle changes in the binding pocket; however, these small changes are associated with an 11 Å outward movement of the cytoplasmic end of transmembrane segment 6, and rearrangements of transmembrane segments 5 and 7 that are remarkably similar to those observed in opsin, an active form of rhodopsin. This structure provides insights into the process of agonist binding and activation.