GPCR-mediated ß-arrestin activation deconvoluted with single-molecule precision.

ß-arrestins bind G protein-coupled receptors to terminate G protein signaling and to facilitate other downstream signaling pathways. Using single-molecule fluorescence resonance energy transfer imaging, we show that ß-arrestin is strongly autoinhibited in its basal state. Its engagement with a phosphopeptide mimicking phosphorylated receptor tail efficiently releases the ß-arrestin tail from its N domain to assume distinct conformations. Unexpectedly, we find that ß-arrestin binding to phosphorylated receptor, with a phosphorylation barcode identical to the isolated phosphopeptide, is highly inefficient and that agonist-promoted receptor activation is required for ß-arrestin activation, consistent with the release of a sequestered receptor C tail. These findings, together with focused cellular investigations, reveal that agonism and receptor C-tail release are specific determinants of the rate and efficiency of ß-arrestin activation by phosphorylated receptor. We infer that receptor phosphorylation patterns, in combination with receptor agonism, synergistically establish the strength and specificity with which diverse, downstream ß-arrestin-mediated events are directed.

GPCR-G Protein-ß-Arrestin Super-Complex Mediates Sustained G Protein Signaling.

Classically, G protein-coupled receptor (GPCR) stimulation promotes G protein signaling at the plasma membrane, followed by rapid ß-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, ß-arrestin, and G protein. These super-complexes or "megaplexes" more readily form at receptors that interact strongly with ß-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 ß-arrestin. The formation of such megaplexes provides a potential physical basis for the newly appreciated sustained G protein signaling from internalized GPCRs.

Signal transduction at GPCRs: Allosteric activation of the ERK MAPK by ß-arrestin.

ß-arrestins are multivalent adaptor proteins that bind active phosphorylated G protein-coupled receptors (GPCRs) to inhibit G protein signaling, mediate receptor internalization, and initiate alternative signaling events. ß-arrestins link agonist-stimulated GPCRs to downstream signaling partners, such as the c-Raf-MEK1-ERK1/2 cascade leading to ERK1/2 activation. ß-arrestins have been thought to transduce signals solely via passive scaffolding by facilitating the assembly of multiprotein signaling complexes. Recently, however, ß-arrestin 1 and 2 were shown to activate two downstream signaling effectors, c-Src and c-Raf, allosterically. Over the last two decades, ERK1/2 have been the most intensely studied signaling proteins scaffolded by ß-arrestins. Here, we demonstrate that ß-arrestins play an active role in allosterically modulating ERK kinase activity in vitro and within intact cells. Specifically, we show that ß-arrestins and their GPCR-mediated active states allosterically enhance ERK2 autophosphorylation and phosphorylation of a downstream ERK2 substrate, and we elucidate the mechanism by which ß-arrestins do so. Furthermore, we find that allosteric stimulation of dually phosphorylated ERK2 by active-state ß-arrestin 2 is more robust than by active-state ß-arrestin 1, highlighting differential capacities of ß-arrestin isoforms to regulate effector signaling pathways downstream of GPCRs. In summary, our study provides strong evidence for a new paradigm in which ß-arrestins function as active "catalytic" scaffolds to allosterically unlock the enzymatic activity of signaling components downstream of GPCR activation.

Mechanism of intracellular allosteric ß2AR antagonist revealed by X-ray crystal structure.

G-protein-coupled receptors (GPCRs) pose challenges for drug discovery efforts because of the high degree of structural homology in the orthosteric pocket, particularly for GPCRs within a single subfamily, such as the nine adrenergic receptors. Allosteric ligands may bind to less-conserved regions of these receptors and therefore are more likely to be selective. Unlike orthosteric ligands, which tonically activate or inhibit signalling, allosteric ligands modulate physiologic responses to hormones and neurotransmitters, and may therefore have fewer adverse effects. The majority of GPCR crystal structures published to date were obtained with receptors bound to orthosteric antagonists, and only a few structures bound to allosteric ligands have been reported. Compound 15 (Cmpd-15) is an allosteric modulator of the ß2 adrenergic receptor (ß2AR) that was recently isolated from a DNA-encoded small-molecule library. Orthosteric ß-adrenergic receptor antagonists, known as beta-blockers, are amongst the most prescribed drugs in the world and Cmpd-15 is the first allosteric beta-blocker. Cmpd-15 exhibits negative cooperativity with agonists and positive cooperativity with inverse agonists. Here we present the structure of the ß2AR bound to a polyethylene glycol-carboxylic acid derivative (Cmpd-15PA) of this modulator. Cmpd-15PA binds to a pocket formed primarily by the cytoplasmic ends of transmembrane segments 1, 2, 6 and 7 as well as intracellular loop 1 and helix 8. A comparison of this structure with inactive- and active-state structures of the ß2AR reveals the mechanism by which Cmpd-15 modulates agonist binding affinity and signalling.

The GPCR-ß-arrestin complex allosterically activates C-Raf by binding its amino terminus.

G protein-coupled receptors (GPCRs) convert external stimuli into cellular signals through heterotrimeric guanine nucleotide-binding proteins (G-proteins) and ß-arrestins (ßarrs). In a ßarr-dependent signaling pathway, ßarrs link GPCRs to various downstream signaling partners, such as the Raf-mitogen-activated protein kinase extracellular signal-regulated kinase-extracellular signal-regulated kinase cascade. Agonist-stimulated GPCR-ßarr complexes have been shown to interact with C-Raf and are thought to initiate the mitogen-activated protein kinase pathway through simple tethering of these signaling partners. However, recent evidence shows that in addition to canonical scaffolding functions, ßarrs can allosterically activate downstream targets, such as the nonreceptor tyrosine kinase Src. Here, we demonstrate the direct allosteric activation of C-Raf by GPCR-ßarr1 complexes in vitro. Furthermore, we show that ßarr1 in complex with a synthetic phosphopeptide mimicking the human V2 vasopressin receptor tail that binds and functionally activates ßarrs also allosterically activates C-Raf. We reveal that the interaction between the phosphorylated GPCR C terminus and ßarr1 is necessary and sufficient for C-Raf activation. Interestingly, the interaction between ßarr1 and C-Raf was considerably reduced in the presence of excess activated H-Ras, a small GTPase known to activate C-Raf, suggesting that H-Ras and ßarr1 bind to the same region on C-Raf. Furthermore, we found that ßarr1 interacts with the Ras-binding domain of C-Raf. Taken together, these data suggest that in addition to canonical scaffolding functions, GPCR-ßarr complexes directly allosterically activate C-Raf by binding to its amino terminus. This work provides novel insights into how ßarrs regulate effector molecules to activate downstream signaling pathways.

Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation.

G-protein-coupled receptors (GPCRs) modulate many physiological processes by transducing a variety of extracellular cues into intracellular responses. Ligand binding to an extracellular orthosteric pocket propagates conformational change to the receptor cytosolic region to promote binding and activation of downstream signalling effectors such as G proteins and ß-arrestins. It is well known that different agonists can share the same binding pocket but evoke unique receptor conformations leading to a wide range of downstream responses ('efficacy'). Furthermore, increasing biophysical evidence, primarily using the ß2-adrenergic receptor (ß2AR) as a model system, supports the existence of multiple active and inactive conformational states. However, how agonists with varying efficacy modulate these receptor states to initiate cellular responses is not well understood. Here we report stabilization of two distinct ß2AR conformations using single domain camelid antibodies (nanobodies)­a previously described positive allosteric nanobody (Nb80) and a newly identified negative allosteric nanobody (Nb60). We show that Nb60 stabilizes a previously unappreciated low-affinity receptor state which corresponds to one of two inactive receptor conformations as delineated by X-ray crystallography and NMR spectroscopy. We find that the agonist isoprenaline has a 15,000-fold higher affinity for ß2AR in the presence of Nb80 compared to the affinity of isoprenaline for ß2AR in the presence of Nb60, highlighting the full allosteric range of a GPCR. Assessing the binding of 17 ligands of varying efficacy to the ß2AR in the absence and presence of Nb60 or Nb80 reveals large ligand-specific effects that can only be explained using an allosteric model which assumes equilibrium amongst at least three receptor states. Agonists generally exert efficacy by stabilizing the active Nb80-stabilized receptor state (R80). In contrast, for a number of partial agonists, both stabilization of R80 and destabilization of the inactive, Nb60-bound state (R60) contribute to their ability to modulate receptor activation. These data demonstrate that ligands can initiate a wide range of cellular responses by differentially stabilizing multiple receptor states.

ß-Arrestin-Biased Allosteric Modulator Potentiates Carvedilol-Stimulated ß Adrenergic Receptor Cardioprotection.

ß 1 adrenergic receptors (ß 1ARs) are central regulators of cardiac function and a drug target for cardiac disease. As a member of the G protein-coupled receptor family, ß 1ARs activate cellular signaling by primarily coupling to Gs proteins to activate adenylyl cyclase, cAMP-dependent pathways, and the multifunctional adaptor-transducer protein ß-arrestin. Carvedilol, a traditional ß-blocker widely used in treating high blood pressure and heart failure by blocking ß adrenergic receptor-mediated G protein activation, can selectively stimulate Gs-independent ß-arrestin signaling of ß adrenergic receptors, a process known as ß-arrestin-biased agonism. Recently, a DNA-encoded small-molecule library screen against agonist-occupied ß 2 adrenergic receptors (ß 2ARs) identified Compound-6 (Cmpd-6) to be a positive allosteric modulator for agonists on ß 2ARs. Intriguingly, it was further discovered that Cmpd-6 is positively cooperative with the ß-arrestin-biased ligand carvedilol at ß 2ARs. Here we describe the surprising finding that at ß 1ARs unlike ß 2ARs, Cmpd-6 is cooperative only with carvedilol and not agonists. Cmpd-6 increases the binding affinity of carvedilol for ß 1ARs and potentiates carvedilol-stimulated, ß-arrestin-dependent ß 1AR signaling, such as epidermal growth factor receptor transactivation and extracellular signal-regulated kinase activation, whereas it does not have an effect on Gs-mediated cAMP generation. In vivo, Cmpd-6 enhances the antiapoptotic, cardioprotective effect of carvedilol in response to myocardial ischemia/reperfusion injury. This antiapoptotic role of carvedilol is dependent on ß-arrestins since it is lost in mice with myocyte-specific deletion of ß-arrestins. Our findings demonstrate that Cmpd-6 is a selective ß-arrestin-biased allosteric modulator of ß 1ARs and highlight its potential clinical utility in enhancing carvedilol-mediated cardioprotection against ischemic injury. SIGNIFICANCE STATEMENT: This study demonstrates the positive cooperativity of Cmpd-6 on ß1ARs as a ß-arrestin-biased positive allosteric modulator. Cmpd-6 selectively enhances the affinity and cellular signaling of carvedilol, a known ß-arrestin-biased ß-blocker for ß1ARs, whereas it has minimal effect on other ligands tested. Importantly, Cmpd-6 enhances the ß-arrestin-dependent in vivo cardioprotective effect of carvedilol during ischemia/reperfusion injury-induced apoptosis. The data support the potential therapeutic application of Cmpd-6 to enhance the clinical benefits of carvedilol in the treatment of cardiac disease.

Unique Positive Cooperativity Between the ß-Arrestin-Biased ß-Blocker Carvedilol and a Small Molecule Positive Allosteric Modulator of the ß2-Adrenergic Receptor.

Among ß-blockers that are clinically prescribed for heart failure, carvedilol is a first-choice agent with unique pharmacological properties. Carvedilol is distinct from other ß-blockers in its ability to elicit ß-arrestin-biased agonism, which has been suggested to underlie its cardioprotective effects. Augmenting the pharmacologic properties of carvedilol thus holds the promise of developing more efficacious and/or biased ß-blockers. We recently identified compound-6 (cmpd-6), the first small molecule positive allosteric modulator of the ß2-adrenergic receptor (ß2AR). Cmpd-6 is positively cooperative with orthosteric agonists at the ß2AR and enhances agonist-mediated transducer (G-protein and ß-arrestin) signaling in an unbiased manner. Here, we report that cmpd-6, quite unexpectedly, displays strong positive cooperativity only with carvedilol among a panel of structurally diverse ß-blockers. Cmpd-6 enhances the binding affinity of carvedilol for the ß2AR and augments its ability to competitively antagonize agonist-induced cAMP generation. Cmpd-6 potentiates ß-arrestin1- but not Gs-protein-mediated high-affinity binding of carvedilol at the ß2AR and ß-arrestin-mediated cellular functions in response to carvedilol including extracellular signal-regulated kinase phosphorylation, receptor endocytosis, and trafficking into lysosomes. Importantly, an analog of cmpd-6 that selectively retains positive cooperativity with carvedilol acts as a negative modulator of agonist-stimulated ß2AR signaling. These unprecedented cooperative properties of carvedilol and cmpd-6 have implications for fundamental understanding of G-protein-coupled receptor (GPCR) allosteric modulation, as well as for the development of more effective biased beta blockers and other GPCR therapeutics. SIGNIFICANCE STATEMENT: This study reports on the small molecule-mediated allosteric modulation of the ß-arrestin-biased ß-blocker, carvedilol. The small molecule, compound-6 (cmpd-6), displays an exclusive positive cooperativity with carvedilol among other ß-blockers and enhances the binding affinity of carvedilol for the ß2-adrenergic receptor. Cooperative effects of cmpd-6 augment the ß-blockade property of carvedilol while potentiating its ß-arrestin-mediated signaling functions. These findings have potential implications in advancing G-protein-coupled receptor allostery, developing biased therapeutics and remedying cardiovascular ailments.

Visualization of arrestin recruitment by a G-protein-coupled receptor.

G-protein-coupled receptors (GPCRs) are critically regulated by ß-arrestins, which not only desensitize G-protein signalling but also initiate a G-protein-independent wave of signalling. A recent surge of structural data on a number of GPCRs, including the ß2 adrenergic receptor (ß2AR)-G-protein complex, has provided novel insights into the structural basis of receptor activation. However, complementary information has been lacking on the recruitment of ß-arrestins to activated GPCRs, primarily owing to challenges in obtaining stable receptor-ß-arrestin complexes for structural studies. Here we devised a strategy for forming and purifying a functional human ß2AR-ß-arrestin-1 complex that allowed us to visualize its architecture by single-particle negative-stain electron microscopy and to characterize the interactions between ß2AR and ß-arrestin 1 using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and chemical crosslinking. Electron microscopy two-dimensional averages and three-dimensional reconstructions reveal bimodal binding of ß-arrestin 1 to the ß2AR, involving two separate sets of interactions, one with the phosphorylated carboxy terminus of the receptor and the other with its seven-transmembrane core. Areas of reduced HDX together with identification of crosslinked residues suggest engagement of the finger loop of ß-arrestin 1 with the seven-transmembrane core of the receptor. In contrast, focal areas of raised HDX levels indicate regions of increased dynamics in both the N and C domains of ß-arrestin 1 when coupled to the ß2AR. A molecular model of the ß2AR-ß-arrestin signalling complex was made by docking activated ß-arrestin 1 and ß2AR crystal structures into the electron microscopy map densities with constraints provided by HDX-MS and crosslinking, allowing us to obtain valuable insights into the overall architecture of a receptor-arrestin complex. The dynamic and structural information presented here provides a framework for better understanding the basis of GPCR regulation by arrestins.

Allosteric "beta-blocker" isolated from a DNA-encoded small molecule library.

The ß2-adrenergic receptor (ß2AR) has been a model system for understanding regulatory mechanisms of G-protein-coupled receptor (GPCR) actions and plays a significant role in cardiovascular and pulmonary diseases. Because all known ß-adrenergic receptor drugs target the orthosteric binding site of the receptor, we set out to isolate allosteric ligands for this receptor by panning DNA-encoded small-molecule libraries comprising 190 million distinct compounds against purified human ß2AR. Here, we report the discovery of a small-molecule negative allosteric modulator (antagonist), compound 15 [([4-((2S)-3-(((S)-3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)amino)-2-(2-cyclohexyl-2-phenylacetamido)-3-oxopropyl)benzamide], exhibiting a unique chemotype and low micromolar affinity for the ß2AR. Binding of 15 to the receptor cooperatively enhances orthosteric inverse agonist binding while negatively modulating binding of orthosteric agonists. Studies with a specific antibody that binds to an intracellular region of the ß2AR suggest that 15 binds in proximity to the G-protein binding site on the cytosolic surface of the ß2AR. In cell-signaling studies, 15 inhibits cAMP production through the ß2AR, but not that mediated by other Gs-coupled receptors. Compound 15 also similarly inhibits ß-arrestin recruitment to the activated ß2AR. This study presents an allosteric small-molecule ligand for the ß2AR and introduces a broadly applicable method for screening DNA-encoded small-molecule libraries against purified GPCR targets. Importantly, such an approach could facilitate the discovery of GPCR drugs with tailored allosteric effects.

Distinct conformations of GPCR-ß-arrestin complexes mediate desensitization, signaling, and endocytosis.

ß-Arrestins (ßarrs) interact with G protein-coupled receptors (GPCRs) to desensitize G protein signaling, to initiate signaling on their own, and to mediate receptor endocytosis. Prior structural studies have revealed two unique conformations of GPCR-ßarr complexes: the "tail" conformation, with ßarr primarily coupled to the phosphorylated GPCR C-terminal tail, and the "core" conformation, where, in addition to the phosphorylated C-terminal tail, ßarr is further engaged with the receptor transmembrane core. However, the relationship of these distinct conformations to the various functions of ßarrs is unknown. Here, we created a mutant form of ßarr lacking the "finger-loop" region, which is unable to form the core conformation but retains the ability to form the tail conformation. We find that the tail conformation preserves the ability to mediate receptor internalization and ßarr signaling but not desensitization of G protein signaling. Thus, the two GPCR-ßarr conformations can carry out distinct functions.

Small-Molecule Positive Allosteric Modulators of the ß2-Adrenoceptor Isolated from DNA-Encoded Libraries.

Conventional drug discovery efforts at the ß2-adrenoceptor (ß2AR) have led to the development of ligands that bind almost exclusively to the receptor's hormone-binding orthosteric site. However, targeting the largely unexplored and evolutionarily unique allosteric sites has potential for developing more specific drugs with fewer side effects than orthosteric ligands. Using our recently developed approach for screening G protein-coupled receptors (GPCRs) with DNA-encoded small-molecule libraries, we have discovered and characterized the first ß2AR small-molecule positive allosteric modulators (PAMs)-compound (Cmpd)-6 [(R)-N-(4-amino-1-(4-(tert-butyl)phenyl)-4-oxobutan-2-yl)-5-(N-isopropyl-N-methylsulfamoyl)-2-((4-methoxyphenyl)thio)benzamide] and its analogs. We used purified human ß2ARs, occupied by a high-affinity agonist, for the affinity-based screening of over 500 million distinct library compounds, which yielded Cmpd-6. It exhibits a low micro-molar affinity for the agonist-occupied ß2AR and displays positive cooperativity with orthosteric agonists, thereby enhancing their binding to the receptor and ability to stabilize its active state. Cmpd-6 is cooperative with G protein and ß-arrestin1 (a.k.a. arrestin2) to stabilize high-affinity, agonist-bound active states of the ß2AR and potentiates downstream cAMP production and receptor recruitment of ß-arrestin2 (a.k.a. arrestin3). Cmpd-6 is specific for the ß2AR compared with the closely related ß1AR. Structure-activity studies of select Cmpd-6 analogs defined the chemical groups that are critical for its biologic activity. We thus introduce the first small-molecule PAMs for the ß2AR, which may serve as a lead molecule for the development of novel therapeutics. The approach described in this work establishes a broadly applicable proof-of-concept strategy for affinity-based discovery of small-molecule allosteric compounds targeting unique conformational states of GPCRs.

Conformationally selective RNA aptamers allosterically modulate the ß2-adrenoceptor.

G-protein-coupled receptor (GPCR) ligands function by stabilizing multiple, functionally distinct receptor conformations. This property underlies the ability of 'biased agonists' to activate specific subsets of a given receptor's signaling profile. However, stabilizing distinct active GPCR conformations to enable structural characterization of mechanisms underlying GPCR activation remains difficult. These challenges have accentuated the need for receptor tools that allosterically stabilize and regulate receptor function through unique, previously unappreciated mechanisms. Here, using a highly diverse RNA library combined with advanced selection strategies involving state-of-the-art next-generation sequencing and bioinformatics analyses, we identify RNA aptamers that bind a prototypical GPCR, the ß2-adrenoceptor (ß2AR). Using biochemical, pharmacological, and biophysical approaches, we demonstrate that these aptamers bind with nanomolar affinity at defined surfaces of the receptor, allosterically stabilizing active, inactive, and ligand-specific receptor conformations. The discovery of RNA aptamers as allosteric GPCR modulators significantly expands the diversity of ligands available to study the structural and functional regulation of GPCRs.

Design, synthesis, and functional assessment of Cmpd-15 derivatives as negative allosteric modulators for the ß2-adrenergic receptor.

The ß2-adrenergic receptor (ß2AR), a G protein-coupled receptor, is an important therapeutic target. We recently described Cmpd-15, the first small molecule negative allosteric modulator (NAM) for the ß2AR. Herein we report in details the design, synthesis and structure-activity relationships (SAR) of seven Cmpd-15 derivatives. Furthermore, we provide in a dose-response paradigm, the details of the effects of these derivatives in modulating agonist-induced ß2AR activities (G-protein-mediated cAMP production and ß-arrestin recruitment to the receptor) as well as the binding affinity of an orthosteric agonist in radio-ligand competition binding assay. Our results show that some modifications, including removal of the formamide group in the para-formamido phenylalanine region and bromine in the meta-bromobenzyl methylbenzamide region caused dramatic reduction in the functional activity of Cmpd-15. These SAR results provide valuable insights into the mechanism of action of the NAM Cmpd-15 as well as the basis for future development of more potent and selective modulators for the ß2AR based on the chemical scaffold of Cmpd-15.

Beta-arrestin 1 mediated Src activation via Src SH3 domain revealed by cryo-electron microscopy.

Beta-arrestins (ßarrs) are key regulators and transducers of G-protein coupled receptor signaling; however, little is known of how ßarrs communicate with their downstream effectors. Here, we use cryo-electron microscopy to elucidate how ßarr1 recruits and activates non-receptor tyrosine kinase Src. ßarr1 binds Src SH3 domain via two distinct sites: a polyproline site in the N-domain and a non-proline site in the central crest region. At both sites ßarr1 interacts with the aromatic surface of SH3 which is critical for Src autoinhibition, suggesting that ßarr1 activates Src by SH3 domain displacement. Binding of SH3 to the central crest region induces structural rearrangements in the ß-strand V, finger, and middle loops of ßarr1 and interferes with ßarr1 coupling to the receptor core potentially impacting receptor desensitization and downstream signaling.

Role of the V2R-ßarrestin-Gßγ complex in promoting G protein translocation to endosomes.

Classically, G protein-coupled receptors (GPCRs) promote signaling at the plasma membrane through activation of heterotrimeric Gαßγ proteins, followed by the recruitment of GPCR kinases and ßarrestin (ßarr) to initiate receptor desensitization and internalization. However, studies demonstrated that some GPCRs continue to signal from internalized compartments, with distinct cellular responses. Both ßarr and Gßγ contribute to such non-canonical endosomal G protein signaling, but their specific roles and contributions remain poorly understood. Here, we demonstrate that the vasopressin V2 receptor (V2R)-ßarr complex scaffolds Gßγ at the plasma membrane through a direct interaction with ßarr, enabling its transport to endosomes. Gßγ subsequently potentiates Gαs endosomal translocation, presumably to regenerate an endosomal pool of heterotrimeric Gs. This work shines light on the mechanism underlying G protein subunits translocation from the plasma membrane to the endosomes and provides a basis for understanding the role of ßarr in mediating sustained G protein signaling.

Multiple ligand-specific conformations of the ß2-adrenergic receptor.

Seven-transmembrane receptors (7TMRs), also called G protein-coupled receptors (GPCRs), represent the largest class of drug targets, and they can signal through several distinct mechanisms including those mediated by G proteins and the multifunctional adaptor proteins ß-arrestins. Moreover, several receptor ligands with differential efficacies toward these distinct signaling pathways have been identified. However, the structural basis and mechanism underlying this 'biased agonism' remains largely unknown. Here, we develop a quantitative mass spectrometry strategy that measures specific reactivities of individual side chains to investigate dynamic conformational changes in the ß(2)-adrenergic receptor occupied by nine functionally distinct ligands. Unexpectedly, only a minority of residues showed reactivity patterns consistent with classical agonism, whereas the majority showed distinct patterns of reactivity even between functionally similar ligands. These findings demonstrate, contrary to two-state models for receptor activity, that there is significant variability in receptor conformations induced by different ligands, which has significant implications for the design of new therapeutic agents.

Allosteric modulator potentiates ß2AR agonist-promoted bronchoprotection in asthma models.

Asthma is a chronic inflammatory disease associated with episodic airway narrowing. Inhaled ß2-adrenergic receptor (ß2AR) agonists (ß2-agonists) promote - with limited efficacy - bronchodilation in asthma. All ß2-agonists are canonical orthosteric ligands that bind the same site as endogenous epinephrine. We recently isolated a ß2AR-selective positive allosteric modulator (PAM), compound-6 (Cmpd-6), which binds outside of the orthosteric site and modulates orthosteric ligand functions. With the emerging therapeutic potential of G-protein coupled receptor allosteric ligands, we investigated the impact of Cmpd-6 on ß2AR-mediated bronchoprotection. Consistent with our findings using human ß2ARs, Cmpd-6 allosterically potentiated ß2-agonist binding to guinea pig ß2ARs and downstream signaling of ß2ARs. In contrast, Cmpd-6 had no such effect on murine ß2ARs, which lack a crucial amino acid in the Cmpd-6 allosteric binding site. Importantly, Cmpd-6 enhanced ß2 agonist-mediated bronchoprotection against methacholine-induced bronchoconstriction in guinea pig lung slices, but - in line with the binding studies - not in mice. Moreover, Cmpd-6 robustly potentiated ß2 agonist-mediated bronchoprotection against allergen-induced airway constriction in lung slices obtained from a guinea pig model of allergic asthma. Cmpd-6 similarly enhanced ß2 agonist-mediated bronchoprotection against methacholine-induced bronchoconstriction in human lung slices. Our results highlight the potential of ß2AR-selective PAMs in the treatment of airway narrowing in asthma and other obstructive respiratory diseases.

G protein-coupled receptor kinase 2 activates radixin, regulating membrane protrusion and motility in epithelial cells.

Ezrin/radixin/moesin (ERM) proteins are membrane-cytoskeleton linkers that also have roles in signal transduction. Here we show that G protein-coupled receptor kinase 2 (GRK2) regulates membrane protrusion and cell migration during wound closure in Madin-Darby canine kidney (MDCK) epithelial cell monolayers at least partly through activating phosphorylation of radixin on a conserved, regulatory C-terminal Thr residue. GRK2 phosphorylated radixin exclusively on Thr 564 in vitro. Expression of a phosphomimetic (Thr-564-to-Asp) mutant of radixin resulted in increased Rac1 activity, membrane protrusion and cell motility in MDCK cells, suggesting that radixin functions "upstream" of Rac1, presumably as a scaffolding protein. Phosphorylation of ERM proteins was highest during the most active phase of epithelial cell sheet migration over the course of wound closure. In view of these results, we explored the mode of action of quinocarmycin/quinocarcin analog DX-52-1, an inhibitor of cell migration and radixin function with considerable selectivity for radixin over the other ERM proteins, finding that its mechanism of inhibition of radixin does not appear to involve binding and antagonism at the site of regulatory phosphorylation.

Noncanonical scaffolding of Gαi and ß-arrestin by G protein-coupled receptors.

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) are common drug targets and canonically couple to specific Gα protein subtypes and ß-arrestin adaptor proteins. G protein-mediated signaling and ß-arrestin-mediated signaling have been considered separable. We show here that GPCRs promote a direct interaction between Gαi protein subtype family members and ß-arrestins regardless of their canonical Gα protein subtype coupling. Gαi:ß-arrestin complexes bound extracellular signal-regulated kinase (ERK), and their disruption impaired both ERK activation and cell migration, which is consistent with ß-arrestins requiring a functional interaction with Gαi for certain signaling events. These results introduce a GPCR signaling mechanism distinct from canonical G protein activation in which GPCRs cause the formation of Gαi:ß-arrestin signaling complexes.