Allosteric Coupling of Drug Binding and Intracellular Signaling in the A2A Adenosine Receptor.

Signaling across cellular membranes, the 826 human G protein-coupled receptors (GPCRs) govern a wide range of vital physiological processes, making GPCRs prominent drug targets. X-ray crystallography provided GPCR molecular architectures, which also revealed the need for additional structural dynamics data to support drug development. Here, nuclear magnetic resonance (NMR) spectroscopy with the wild-type-like A2A adenosine receptor (A2AAR) in solution provides a comprehensive characterization of signaling-related structural dynamics. All six tryptophan indole and eight glycine backbone 15N-1H NMR signals in A2AAR were individually assigned. These NMR probes provided insight into the role of Asp522.50 as an allosteric link between the orthosteric drug binding site and the intracellular signaling surface, revealing strong interactions with the toggle switch Trp 2466.48, and delineated the structural response to variable efficacy of bound drugs across A2AAR. The present data support GPCR signaling based on dynamic interactions between two semi-independent subdomains connected by an allosteric switch at Asp522.50.

Restructuring G-protein- coupled receptor activation.

G-protein-coupled receptors serve as key signal transduction conduits, linking extracellular inputs with diverse cellular responses. These receptors eluded structural characterization for decades following their identification. A landmark structure of rhodopsin provided a basis for structure-function studies and homology modeling, but advances in receptor biology suffered from a lack of receptor-specific structural insights. The recent explosion in GPCR structures confirms some features predicted by rhodopsin-based models, and more importantly, it reveals unexpected ligand-binding modes and critical aspects of the receptor activation process. The new structures also promise to foster studies testing emerging models for GPCR function such as receptor dimerization and ligand-biased signaling.

Crystal structure of misoprostol bound to the labor inducer prostaglandin E2 receptor.

Misoprostol is a life-saving drug in many developing countries for women at risk of post-partum hemorrhaging owing to its affordability, stability, ease of administration and clinical efficacy. However, misoprostol lacks receptor and tissue selectivities, and thus its use is accompanied by a number of serious side effects. The development of pharmacological agents combining the advantages of misoprostol with improved selectivity is hindered by the absence of atomic details of misoprostol action in labor induction. Here, we present the 2.5 Å resolution crystal structure of misoprostol free-acid form bound to the myometrium labor-inducing prostaglandin E2 receptor 3 (EP3). The active state structure reveals a completely enclosed binding pocket containing a structured water molecule that coordinates misoprostol's ring structure. Modeling of selective agonists in the EP3 structure reveals rationales for selectivity. These findings will provide the basis for the next generation of uterotonic drugs that will be suitable for administration in low resource settings.

Publisher Correction: Crystal structure of misoprostol bound to the labor inducer prostaglandin E2 receptor.

In the version of this article originally published, the present address for Petr Popov was incorrectly listed as 'Koltech Institute of Science & Technology, Moscow, Russia'. The correct present address is 'Skolkovo Institute of Science and Technology, Moscow, Russia'. The error has been corrected in the HTML and PDF versions of the paper.

A systematic analysis of prostaglandin E2 type 3 receptor isoform signaling reveals isoform- and species-dependent L798106 Gαz-biased agonist responses.

Prostaglandins are bioactive lipids involved in many physiological and pathophysiological conditions, such as pain, atherosclerosis, type II diabetes, and parturition. Prostaglandin E2 (PGE2) activates four G protein-coupled receptors (GPCRs), named the PGE2 types 1-4 receptors (EP1-4), to elicit the intracellular signaling responsible for their physiological actions. There are more than twelve EP3 isoforms in humans that differ only by the sequence of their C-termini. However, the signaling mechanisms engaged by the various isoforms have never been clearly defined. In this study, we used a recently described BRET-based biosensor technology to define the signaling profiles for each of the human isoforms on a selection of signaling pathways using the agonists, PGE2 and sulprostone, and the purportedly EP3-specific antagonist L798106. We found that L798106 is a biased agonist of the Gαz pathway for some human EP3 isoforms, an effect that is not detected in the close ortholog mouse EP3 isoform α. We also found that the presence of a threonine residue at position 107 in the binding site of human EP3, which is a serine in most other species including mice, is important for L798106-mediated Gαz efficacy. Given the reported importance of EP3-Gαz signaling on the potential therapeutic efficacy of EP3 and since many preclinical studies for these mechanisms have been performed in rodents, this finding demonstrates the importance of determining a detailed signaling profile of ligands for different species and receptor isoforms, which constitutes an important step to better understand the therapeutic potential of the EP3.

Protein-protein interactions monitored in cells from transgenic mice using bioluminescence resonance energy transfer.

Monitoring the dynamics of protein-protein interactions in their natural environment remains a challenge. Resonance energy transfer approaches represent a promising avenue to directly probe these interactions in real time. The present study aims at establishing a proof of principle that bioluminescence resonance energy transfer (BRET) can be used to study the regulation of protein-protein interaction in cells from transgenic animals. A transgenic mouse line coexpressing the beta(2)-adrenergic receptor fused to Renilla luciferase (beta(2)AR-Rluc) and beta arrestin-2 fused to a green fluorescent protein (GFP2-beta arr2) was generated. The fusion proteins were found to be functional in the transgenic animals and the beta(2)AR-Rluc maintained pharmacological properties, comparable to that of the native receptor. Sufficiently high luminescence signal was generated to allow detection of BRET in testis cells where the beta(2)AR-Rluc transgene was expressed at levels significantly higher than that of the endogenous receptor in this tissue but remain within physiological range when compared with other beta(2)AR-expressing tissues. Stimulation with a beta-adrenergic agonist led to a significant dose- and time-dependent increase in BRET, which reflected ligand-promoted recruitment of beta arr2 to the receptor. Our study demonstrates that BRET can be used to monitor the dynamic regulation of protein-protein interactions in cells derived from transgenic mice.

Probing the activation-promoted structural rearrangements in preassembled receptor-G protein complexes.

Activation of heterotrimeric G proteins by their cognate seven transmembrane domain receptors is believed to involve conformational changes propagated from the receptor to the G proteins. However, the nature of these changes remains unknown. We monitored the conformational rearrangements at the interfaces between receptors and G proteins and between G protein subunits by measuring bioluminescence resonance energy transfer between probes inserted at multiple sites in receptor-G protein complexes. Using the data obtained for the alpha(2A)AR-G alpha(i1) beta1gamma2 complex and the available crystal structures of G alpha(i1) beta1gamma2, we propose a model wherein agonist binding induces conformational reorganization of a preexisting receptor-G protein complex, leading the G alpha-G betagamma interface to open but not dissociate. This conformational change may represent the movement required to allow nucleotide exit from the G alpha subunit, thus reflecting the initial activation event.

Insights into signaling from the beta2-adrenergic receptor structure.

With more than 800 members, the G protein-coupled receptor family constitutes the largest group of membrane proteins involved in signal transduction. Until the end of last year, high-resolution three-dimensional structures were available for only one of them--the light receptor rhodopsin. Recently the structure of the beta(2)-adrenergic receptor has been obtained, and it revealed interesting differences with the structure of rhodopsin. Analyses of these differences raise important questions about the binding modes of diffusible ligands in the receptor and allow formulation of testable hypotheses about the structural determinants linking drug binding to specific signaling responses. The three-dimensional structure derived from the beta(2)-adrenergic receptor crystal has been used to virtually dock ligands with distinct activities. The different binding modes of these ligands, which correlated with their reported efficacy profiles, suggest that it could be possible to predict the structural determinants of drug signaling efficacies.

Small-scale approach for precrystallization screening in GPCR X-ray crystallography.

G protein-coupled receptors (GPCRs) are important pharmaceutical targets. Knowledge of their 3D structures is critical to understanding mechanisms of drug action. Low cellular expression, purification yield, and in vitro instability are substantial hurdles to the successful determination of GPCR structure. Intense effort is required to optimize a receptor's protein sequence and purification procedure, increasing the complexity of the precrystallization process. Here, we present a procedure for a small-scale precrystallization screen that involves detecting GPCR expression levels in Spodoptera frugiperda (Sf9) culture by flow cytometry and evaluating GPCR stability by size-exclusion chromatography and UV absorbance measurements. The example procedure uses the smallest volumes of Sf9 cell culture that will yield sufficient quantities of purified protein for intrinsic UV absorbance analysis and is amenable to medium throughput with the same constructs and conditions that would be scaled up for crystallization trials. The protocol takes 8 d to complete and requires expertise in cell culture, protein purification, and chromatography.

Emerging structural biology of lipid G protein-coupled receptors.

The first crystal structure of a G protein-coupled receptor (GPCR) was that of the bovine rhodopsin, solved in 2000, and is a light receptor within retina rode cells that enables vision by transducing a conformational signal from the light-induced isomerization of retinal covalently bound to the receptor. More than 7 years after this initial discovery and following more than 20 years of technological developments in GPCR expression, stabilization, and crystallography, the high-resolution structure of the adrenaline binding ß2 -adrenergic receptor, a ligand diffusible receptor, was discovered. Since then, high-resolution structures of more than 53 unique GPCRs have been determined leading to a significant improvement in our understanding of the basic mechanisms of ligand-binding and ligand-mediated receptor activation that revolutionized the field of structural molecular pharmacology of GPCRs. Recently, several structures of eight unique lipid-binding receptors, one of the most difficult GPCR families to study, have been reported. This review presents the outstanding structural and pharmacological features that have emerged from these new lipid receptor structures. The impact of these findings goes beyond mechanistic insights, providing evidence of the fundamental role of GPCRs in the physiological integration of the lipid signaling system, and highlighting the importance of sustained research into the structural biology of GPCRs for the development of new therapeutics targeting lipid receptors.

Subcellular imaging of dynamic protein interactions by bioluminescence resonance energy transfer.

Despite the fact that numerous studies suggest the existence of receptor multiprotein complexes, visualization and monitoring of the dynamics of such protein assemblies remain a challenge. In this study, we established appropriate conditions to consider spatiotemporally resolved images of such protein assemblies using bioluminescence resonance energy transfer (BRET) in mammalian living cells. Using covalently linked Renilla luciferase and yellow fluorescent proteins, we depicted the time course of dynamic changes in the interaction between the V2-vasopressin receptor and beta-arrestin induced by a receptor agonist. The protein-protein interactions were resolved at the level of subcellular compartments (nucleus, plasma membrane, or endocytic vesicules) and in real time within tens-of-seconds to tens-of-minutes time frame. These studies provide a proof of principle as well as experimental parameters and controls required for high-resolution dynamic studies using BRET imaging in single cells.

Evolutionary action and structural basis of the allosteric switch controlling ß2AR functional selectivity.

Functional selectivity of G-protein-coupled receptors is believed to originate from ligand-specific conformations that activate only subsets of signaling effectors. In this study, to identify molecular motifs playing important roles in transducing ligand binding into distinct signaling responses, we combined in silico evolutionary lineage analysis and structure-guided site-directed mutagenesis with large-scale functional signaling characterization and non-negative matrix factorization clustering of signaling profiles. Clustering based on the signaling profiles of 28 variants of the ß2-adrenergic receptor reveals three clearly distinct phenotypical clusters, showing selective impairments of either the Gi or ßarrestin/endocytosis pathways with no effect on Gs activation. Robustness of the results is confirmed using simulation-based error propagation. The structural changes resulting from functionally biasing mutations centered around the DRY, NPxxY, and PIF motifs, selectively linking these micro-switches to unique signaling profiles. Our data identify different receptor regions that are important for the stabilization of distinct conformations underlying functional selectivity.

A new inhibitor of the ß-arrestin/AP2 endocytic complex reveals interplay between GPCR internalization and signalling.

In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, ß-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of ß-arrestin recruitment to the receptor and ß-arrestin-promoted endocytosis in propagating receptor signalling has been limited by the lack of selective analytical tools. Here, using a combination of virtual screening and cell-based assays, we have identified a small molecule that selectively inhibits the interaction between ß-arrestin and the ß2-adaptin subunit of the clathrin adaptor protein AP2 without interfering with the formation of receptor/ß-arrestin complexes. This selective ß-arrestin/ß2-adaptin inhibitor (Barbadin) blocks agonist-promoted endocytosis of the prototypical ß2-adrenergic (ß2AR), V2-vasopressin (V2R) and angiotensin-II type-1 (AT1R) receptors, but does not affect ß-arrestin-independent (transferrin) or AP2-independent (endothelin-A) receptor internalization. Interestingly, Barbadin fully blocks V2R-stimulated ERK1/2 activation and blunts cAMP accumulation promoted by both V2R and ß2AR, supporting the concept of ß-arrestin/AP2-dependent signalling for both G protein-dependent and -independent pathways.

High-throughput screening of G protein-coupled receptor antagonists using a bioluminescence resonance energy transfer 1-based beta-arrestin2 recruitment assay.

In this study, the authors developed HEK293 cell lines that stably coexpressed optimal amounts of beta-arrestin2-Rluc and VENUS fusions of G protein-coupled receptors (GPCRs) belonging to both class A and class B receptors, which include receptors that interact transiently or stably with beta-arrestins. This allowed the use of a bioluminescence resonance energy transfer (BRET) 1- beta-arrestin2 translocation assay to quantify receptor activation or inhibition. One of the developed cell lines coexpressing CCR5-VENUS and beta-arrestin2- Renilla luciferase was then used for high-throughput screening (HTS) for antagonists of the chemokine receptor CCR5, the primary co-receptor for HIV. A total of 26,000 compounds were screened for inhibition of the agonist-promoted beta-arrestin2 recruitment to CCR5, and 12 compounds were found to specifically inhibit the agonist-induced beta-arrestin2 recruitment to CCR5. Three of the potential hits were further tested using other functional assays, and their abilities to inhibit CCR5 agonist-promoted signaling were confirmed. This is the 1st study describing a BRET1-beta-arrestin recruitment assay in stable mammalian cells and its successful application in HTS for GPCRs antagonists.

Impedance responses reveal ß2-adrenergic receptor signaling pluridimensionality and allow classification of ligands with distinct signaling profiles.

The discovery that drugs targeting a single G protein-coupled receptor (GPCR) can differentially modulate distinct subsets of the receptor signaling repertoire has created a challenge for drug discovery at these important therapeutic targets. Here, we demonstrate that a single label-free assay based on cellular impedance provides a real-time integration of multiple signaling events engaged upon GPCR activation. Stimulation of the ß2-adrenergic receptor (ß2AR) in living cells with the prototypical agonist isoproterenol generated a complex, multi-featured impedance response over time. Selective pharmacological inhibition of specific arms of the ß2AR signaling network revealed the differential contribution of G(s)-, G(i)- and Gßγ-dependent signaling events, including activation of the canonical cAMP and ERK1/2 pathways, to specific components of the impedance response. Further dissection revealed the essential role of intracellular Ca²âº in the impedance response and led to the discovery of a novel ß2AR-promoted Ca²âº mobilization event. Recognizing that impedance responses provide an integrative assessment of ligand activity, we screened a collection of ß-adrenergic ligands to determine if differences in the signaling repertoire engaged by compounds would lead to distinct impedance signatures. An unsupervised clustering analysis of the impedance responses revealed the existence of 5 distinct compound classes, revealing a richer signaling texture than previously recognized for this receptor. Taken together, these data indicate that the pluridimensionality of GPCR signaling can be captured using integrative approaches to provide a comprehensive readout of drug activity.