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Tetrahydrofuran-containing clathrate hydrate is considered as a potential cold and very cold neutron moderator material. The fully deuterated form is more promising because of the lower neutron absorption cross section of deuterium. In this work we present theoretical calculations of neutron scattering cross sections for hydrogenated and deuterated clathrates at low temperature. The scattering cross sections are generated by using the crystalline structure and phonon density of states obtained from density functional theory calculations. The theoretical neutron scattering cross sections serve to compare against existing or future experimental data.

Background: Ang-(1-7) (angiotensin (1-7)) via MasR (Mas receptor) opposes vaso-injurious actions of Ang II (angiotensin II) as shown in models of pulmonary hypertension. The underlying mechanisms remain unclear. We hypothesized cross talk between Ang-(1-7) and the protective arm of the ET-1 (endothelin-1) system involving MasR and ET B R (endothelin receptor type B)., Methods: To address this, we studied multiple models: in vivo, in a mouse model of ET-1-associated vascular injury (hypoxia-induced pulmonary hypertension); ex vivo, in isolated mouse arteries; and in vitro, in human endothelial cells., Results: Pulmonary hypertension mice exhibited pulmonary vascular remodeling, endothelial dysfunction, and ET-1-induced hypercontractility. Ang-(1-7) treatment (14 days) ameliorated these effects and increased the expression of vascular ET B R. In human endothelial cells, Ang-(1-7)-induced activation of eNOS (endothelial NO synthase)/NO was attenuated by A779 (MasR antagonist) and BQ788 (ET B R antagonist). A779 inhibited ET-1-induced signaling. Coimmunoprecipitation and peptide array experiments demonstrated the interaction between MasR and ET B R. Binding sites for ET B R were mapped to MasR (amino acids 290-314). Binding sites for MasR on ET B R were identified (amino acids 176-200). Peptides that disrupt MasR:ET B R prevented Ang-(1-7) and ET-1 signaling. Using high-throughput screening, we identified compounds that enhance MasR:ET B R interaction, which we termed enhancers. Enhancers increased Ang-(1-7)-induced eNOS activity, NO production, and Ang-(1-7)-mediated vasorelaxation, and reduced contractile responses., Conclusions: We identify cross talk between Ang-(1-7) and ET-1 through MasR:ET B R interaction as a novel network that is vasoprotective. Promoting coactivity between these systems amplifies Ang-(1-7) signaling, increases ET-1/ET B R-mediated vascular actions, and attenuates the injurious effects of ET-1. Enhancing Ang-(1-7)/MasR:ET-1/ET B R signaling may have therapeutic potential in conditions associated with vascular damage., Competing Interests: None.

The increasing shift from cannabis smoking to cannabis vaping is largely driven by the perception that vaping to form an aerosol represents a safer alternative to smoking and is a form of consumption appealing to youth. Herein, we compared the chemical composition and receptor-mediated activity of cannabis smoke extract (CaSE) to cannabis vaping extract (CaVE) along with the biological response in human bronchial epithelial cells. Chemical analysis using HPLC and GC/MS revealed that cannabis vaping aerosol contained fewer toxicants than smoke; CaSE and CaVE contained teratogens, carcinogens, and respiratory toxicants. A bioluminescence resonance energy transfer (BRET)-based biosensor detected the receptor-mediated activity of the extracts, primarily driven by Δ9-THC concentration. RNA- sequencing showed both CaSE and CaVE induced similar transcriptional responses, significantly upregulating genes within pathways related to inflammation, cancer, and cellular stress. This was paralleled by downregulation of pathways related to lipid synthesis and metabolism similarly from both CaSE and CaVE. Targeted metabolomics revealed significant changes in metabolites involved in lipid and membrane metabolism, energy production, nucleotide/DNA/RNA pathways, and oxidative stress response, suggesting potential impairment of lung epithelial cell repair and function. Additionally, the upregulation of 5-hydroxymethylcytosine (5hmC) indicates epigenetic changes potentially contributing to inflammation, oxidative stress, and an increased risk of cancer. These findings challenge the notion that cannabis vaping is risk-free, highlighting an urgent need for comprehensive research into its respiratory health effects. This comparison of cannabis consumption methods offers insights that could inform public health policies and raise consumer awareness regarding the potential risks of inhaling cannabis aerosol.

Inherited or sporadic loss of the TSC2 gene can lead to pulmonary lymphangioleiomyomatosis (LAM), a rare cystic lung disease caused by protease-secreting interstitial tumor nodules. The nodules arise by metastasis of cells that exhibit features of neural crest and smooth muscle lineage ('LAM cells'). Their aberrant growth is attributed to increased activity of 'mechanistic target of rapamycin complex 1' (mTORC1), an anabolic protein kinase that is normally suppressed by the TSC1-TSC2 protein complex. The mTORC1 inhibitor rapamycin slows the progression of LAM, but fails to eradicate disease, indicating a role for mTORC1-independent mechanisms in LAM pathogenesis. Our previous studies revealed G-protein coupled urotensin-II receptor (UT) signaling as a candidate mechanism, but how it promotes oncogenic signaling in TSC2 -deficient cells remained unknown. Using a human pluripotent stem cell-derived in vitro model of LAM, we now show hyperactivation of UT, which was required for their enhanced migration and pro-neoplastic signaling in a rapamycin-insensitive mechanism that required heterotrimeric Gαq/11 (Gαq). Bioluminescence resonance energy transfer assays in HEK 293T cells lacking TSC2 demonstrated selective and enhanced activation of Gαq and its RhoA-associated effectors compared to wild-type control cells. By immunoprecipitation, recombinant UT was physically associated with Gαq and TSC2. The augmented Gαq signaling in TSC2 -deleted cells was independent of mTOR activity, and associated with increased endosomal targeting of p63RhoGEF, a known RhoA-activating effector of Gαq. These studies identify potential mTORC1-independent pro-neoplastic mechanisms that can be targeted for prevention or eradication of pulmonary and extrapulmonary LAM tumors.

Chemokine receptors constitute an important subfamily of G protein-coupled receptors (GPCRs), and they are critically involved in a broad range of immune response mechanisms. Ligand promiscuity among these receptors makes them an interesting target to explore multiple aspects of biased agonism. Here, we comprehensively characterize two chemokine receptors namely, CXCR4 and CXCR7, in terms of their transducer-coupling and downstream signaling upon their stimulation by a common chemokine agonist, CXCL12, and a small molecule agonist, VUF11207. We observe that CXCR7 lacks G-protein-coupling while maintaining robust βarr recruitment with a major contribution of GRK5/6. On the other hand, CXCR4 displays robust G-protein activation as expected but exhibits significantly reduced βarr-coupling compared to CXCR7. These two receptors induce distinct βarr conformations even when activated by the same agonist, and CXCR7, unlike CXCR4, fails to activate ERK1/2 MAP kinase. We also identify a key contribution of a single phosphorylation site in CXCR7 for βarr recruitment and endosomal localization. Our study provides molecular insights into intrinsic-bias encoded in the CXCR4-CXCR7 system with broad implications for drug discovery., (© 2023. Springer Nature Limited.)

G protein-coupled receptors engage both G proteins and β-arrestins, and their coupling can be biased by ligands and mutations. Here, to resolve structural elements and mechanisms underlying effector coupling to the angiotensin II (AngII) type 1 receptor (AT1R), we combined alanine scanning mutagenesis of the entire sequence of the receptor with pharmacological profiling of Gα q and β-arrestin engagement to mutant receptors and molecular dynamics simulations. We showed that Gα q coupling to AT1R involved a large number of residues spread across the receptor, whereas fewer structural regions of the receptor contributed to β-arrestin coupling regulation. Residue stretches in transmembrane domain 4 conferred β-arrestin bias and represented an important structural element in AT1R for functional selectivity. Furthermore, we identified allosteric small-molecule binding sites that were enclosed by communities of residues that produced biased signaling when mutated. Last, we showed that allosteric communication within AT1R emanating from the Gα q coupling site spread beyond the orthosteric AngII-binding site and across different regions of the receptor, including currently unresolved structural regions. Our findings reveal structural elements and mechanisms within AT1R that bias Gα q and β-arrestin coupling and that could be harnessed to design biased receptors for research purposes and to develop allosteric modulators.

Recent studies have shown that G protein coupled receptors (GPCRs) show selective and promiscuous coupling to different Gα protein subfamilies and yet the mechanisms of the range of coupling preferences remain unclear. Here, we use Molecular Dynamics (MD) simulations on ten GPCR:G protein complexes and show that the location (spatial) and duration (temporal) of intermolecular contacts at the GPCR:Gα protein interface play a critical role in how GPCRs selectively interact with G proteins. We identify that some GPCR:G protein interface contacts are common across Gα subfamilies and others specific to Gα subfamilies. Using large scale data analysis techniques on the MD simulation snapshots we derive a spatio-temporal code for contacts that confer G protein selective coupling and validated these contacts using G protein activation BRET assays. Our results demonstrate that promiscuous GPCRs show persistent sampling of the common contacts more than G protein specific contacts. These findings suggest that GPCRs maintain contact with G proteins through a common central interface, while the selectivity comes from G protein specific contacts at the periphery of the interface., (© 2022. The Author(s).)

Promiscuous G protein-coupled receptors (GPCRs) engage multiple Gα subtypes with different efficacies to propagate signals in cells. A mechanistic understanding of Gα selectivity by GPCRs is critical for therapeutic design, since signaling can be restrained by ligand-receptor complexes to preferentially engage specific G proteins. However, details of GPCR selectivity are unresolved. Here, we investigated cognate G protein selectivity using the prototypical promiscuous Gαq/11 and Gα12/13 coupling receptors, angiotensin II type I receptor (AT1R) and prostaglandin F2α receptor (FP), bioluminescence resonance energy transfer-based G protein and pathway-selective sensors, and G protein knockout cells. We determined that competition between G proteins for receptor binding occurred in a receptor- and G protein-specific manner for AT1R and FP but not for other receptors tested. In addition, we show that while Gα12/13 competes with Gαq/11 for AT1R coupling, the opposite occurs for FP, and Gαq-mediated signaling regulated G protein coupling only at AT1R. In cells, the functional modulation of biased ligands at FP and AT1R was contingent upon cognate Gα availability. The efficacy of AT1R-biased ligands, which poorly signal through Gαq/11, increased in the absence of Gα12/13. Finally, we show that a positive allosteric modulator of Gαq/11 signaling that also allosterically decreases FP-Gα12/13 coupling, lost its negative modulation in the absence of Gαq/11 coupling to FP. Together, our findings suggest that despite preferential binding of similar subsets of G proteins, GPCRs follow distinct selectivity rules, which may contribute to the regulation of ligand-mediated G protein bias of AT1R and FP., Competing Interests: Conflict of interest Some of the BRET biosensors used in the present study are licensed to Domain Therapeutics for commercial use. The biosensors are freely available under material transfer agreement for academic research and can be requested from S. A. L. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a primary determinant of β-arrestin (βarr) recruitment and trafficking. For several GPCRs such as the vasopressin receptor subtype 2 (V 2 R), agonist-stimulation first drives the translocation of βarrs to the plasma membrane, followed by endosomal trafficking, which is generally considered to be orchestrated by multiple phosphorylation sites. We have previously shown that mutation of a single phosphorylation site in the V 2 R (i.e., V 2 R T360A ) results in near-complete loss of βarr translocation to endosomes despite robust recruitment to the plasma membrane, and compromised ERK1/2 activation. Here, we discover that a synthetic intrabody (Ib30), which selectively recognizes activated βarr1, efficiently rescues the endosomal trafficking of βarr1 and ERK1/2 activation for V 2 R T360A . Molecular dynamics simulations reveal that Ib30 enriches active-like βarr1 conformation with respect to the inter-domain rotation, and cellular assays demonstrate that it also enhances βarr1-β 2 -adaptin interaction. Our data provide an experimental framework to positively modulate the receptor-transducer-effector axis for GPCRs using intrabodies, which can be potentially integrated in the paradigm of GPCR-targeted drug discovery., (© 2022. The Author(s).)

Cannabis (marijuana) is the most commonly used illicit product in the world and is the second most smoked plant after tobacco. There has been a rapid increase in the number of countries legalizing cannabis for both recreational and medicinal purposes. Smoking cannabis in the form of a joint is the most common mode of cannabis consumption. Combustion of cannabis smoke generates many of the same chemicals as tobacco smoke. Although the impact of tobacco smoke on respiratory health is well-known, the consequence of cannabis smoke on the respiratory system and, in particular, the inflammatory response is unclear. Besides the combustion products present in cannabis smoke, cannabis also contains cannabinoids including Δ 9 -tetrahydrocannabinol (Δ 9 -THC) and cannabidiol (CBD). These compounds are hydrophobic and not present in aqueous solutions. In order to understand the impact of cannabis smoke on pathological mechanisms associated with adverse respiratory outcomes, the development of in vitro surrogates of cannabis smoke exposure is needed. Therefore, we developed a standardized protocol for the generation of cannabis smoke extract (CaSE) to investigate its effect on cellular mechanisms in vitro . First, we determined the concentration of Δ 9 -THC, one of the major cannabinoids, by ELISA and found that addition of methanol to the cell culture media during generation of the aqueous smoke extract significantly increased the amount of Δ 9 -THC. We also observed by LC-MS/MS that CaSE preparation with methanol contains CBD. Using a functional assay in cells for CB1 receptors, the major target of cannabinoids, we found that this CaSE contains Δ 9 -THC which activates CB1 receptors. Finally, this standardized preparation of CaSE induces an inflammatory response in human lung fibroblasts. This study provides an optimized protocol for aqueous CaSE preparation containing biologically active cannabinoids that can be used for in vitro experimentation of cannabis smoke and its potential impact on various indices of pulmonary health., Competing Interests: The Rho BRET biosensor has been licensed to Domain Therapeutics for commercialization. It can be obtained for academic research with a standard academic material transfer agreement (MTA) from SL. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Aloufi, Namkung, Traboulsi, Wilson, Laporte, Kaplan, Ross, Nair, Eidelman and Baglole.)

G-protein-coupled receptors (GPCRs), also known as seven transmembrane receptors (7TMRs), typically interact with two distinct signal-transducers, i.e., G proteins and β-arrestins (βarrs). Interestingly, there are some non-canonical 7TMRs that lack G protein coupling but interact with βarrs, although an understanding of their transducer coupling preference, downstream signaling, and structural mechanism remains elusive. Here, we characterize two such non-canonical 7TMRs, namely, the decoy D6 receptor (D6R) and the complement C5a receptor subtype 2 (C5aR2), in parallel with their canonical GPCR counterparts. We discover that D6R and C5aR2 efficiently couple to βarrs, exhibit distinct engagement of GPCR kinases (GRKs), and activate non-canonical downstream signaling pathways. We also observe that βarrs adopt distinct conformations for D6R and C5aR2, compared to their canonical GPCR counterparts, in response to common natural agonists. Our study establishes D6R and C5aR2 as βarr-coupled 7TMRs and provides key insights into their regulation and signaling with direct implication for biased agonism., Competing Interests: Declaration of interests The authors have no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

ONC201 is a first-in-class imipridone compound that is in clinical trials for the treatment of high-grade gliomas and other advanced cancers. Recent studies identified that ONC201 antagonizes D2-like dopamine receptors at therapeutically relevant concentrations. In the current study, characterization of ONC201 using radioligand binding and multiple functional assays revealed that it was a full antagonist of the D2 and D3 receptors (D2R and D3R) with low micromolar potencies, similar to its potency for antiproliferative effects. Curve-shift experiments using D2R-mediated β -arrestin recruitment and cAMP assays revealed that ONC201 exhibited a mixed form of antagonism. An operational model of allostery was used to analyze these data, which suggested that the predominant modulatory effect of ONC201 was on dopamine efficacy with little to no effect on dopamine affinity. To investigate how ONC201 binds to the D2R, we employed scanning mutagenesis coupled with a D2R-mediated calcium efflux assay. Eight residues were identified as being important for ONC201's functional antagonism of the D2R. Mutation of these residues followed by assessing ONC201 antagonism in multiple signaling assays highlighted specific residues involved in ONC201 binding. Together with computational modeling and simulation studies, our results suggest that ONC201 interacts with the D2R in a bitopic manner where the imipridone core of the molecule protrudes into the orthosteric binding site, but does not compete with dopamine, whereas a secondary phenyl ring engages an allosteric binding pocket that may be associated with negative modulation of receptor activity. SIGNIFICANCE STATEMENT: ONC201 is a novel antagonist of the D2 dopamine receptor with demonstrated efficacy in the treatment of various cancers, especially high-grade glioma. This study demonstrates that ONC201 antagonizes the D2 receptor with novel bitopic and negative allosteric mechanisms of action, which may explain its high selectivity and some of its clinical anticancer properties that are distinct from other D2 receptor antagonists widely used for the treatment of schizophrenia and other neuropsychiatric disorders., (U.S. Government work not protected by U.S. copyright.)

Internalization and intracellular trafficking of G protein-coupled receptors (GPCRs) play pivotal roles in cell responsiveness. Dysregulation in receptor trafficking can lead to aberrant signaling and cell behavior. Here, using an endosomal BRET-based assay in a high-throughput screen with the prototypical GPCR angiotensin II type 1 receptor (AT1R), we sought to identify receptor trafficking inhibitors from a library of ~115,000 small molecules. We identified a novel dual Ras and ARF6 inhibitor, which we named Rasarfin, that blocks agonist-mediated internalization of AT1R and other GPCRs. Rasarfin also potently inhibits agonist-induced ERK1/2 signaling by GPCRs, and MAPK and Akt signaling by EGFR, as well as prevents cancer cell proliferation. In silico modeling and in vitro studies reveal a unique binding modality of Rasarfin within the SOS-binding domain of Ras. Our findings unveil a class of dual small G protein inhibitors for receptor trafficking and signaling, useful for the inhibition of oncogenic cellular responses., (© 2021. The Author(s).)

The angiotensin II (AngII) type 1 receptor (AT1R), a member of the G protein-coupled receptor (GPCR) family, signals through G proteins and β-arrestins, which act as adaptors to regulate AT1R internalization and mitogen-activated protein kinase (MAPK) ERK1/2 activation. β-arrestin-dependent ERK1/2 regulation is the subject of important studies because its spatiotemporal control remains poorly understood for many GPCRs, including AT1R. To study the link between β-arrestin-dependent trafficking and ERK1/2 signaling, we investigated three naturally occurring AT1R variants that show distinct receptor-β-arrestin interactions: A163T, T282M, and C289W. Using bioluminescence resonance energy transfer (BRET)-based and conformational fluorescein arsenical hairpin-BRET sensors coupled with high-resolution fluorescence microscopy, we show that all AT1R variants form complexes with β-arrestin2 at the plasma membrane and efficiently internalize into endosomes upon AngII stimulation. However, mutant receptors imposed distinct conformations in β-arrestin2 and differentially impacted endosomal trafficking and MAPK signaling. Notably, T282M accumulated in endosomes, but its ability to form stable complexes following internalization was reduced, markedly impairing its ability to co-traffic with β-arrestin2. We also found that despite β-arrestin2 overexpression, T282M's and C289W's residency with β-arrestin2 in endosomes was greatly reduced, leading to decreased β-arrestin-dependent ERK1/2 activation, faster recycling of receptors to the plasma membrane, and impaired AngII-mediated proliferation. Our findings reveal that naturally occurring AT1R variants alter the patterns of receptor/β-arrestin2 trafficking and suggest conformationally dependent β-arrestin-mediated MAPK activation as well as endosomal receptor-β-arrestin complex stabilization in the mitogenic response of AT1R., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Cao et al.)

β-arrestins (βarrs) are key regulators of G protein-coupled receptor (GPCR) signaling and trafficking, and their knockdown typically leads to a decrease in agonist-induced ERK1/2 MAP kinase activation. Interestingly, for some GPCRs, knockdown of βarr1 augments agonist-induced ERK1/2 phosphorylation although a mechanistic basis for this intriguing phenomenon is unclear. Here, we use selected GPCRs to explore a possible correlation between the spatial positioning of receptor phosphorylation sites and the contribution of βarr1 in ERK1/2 activation. We discover that engineering a spatially positioned double-phosphorylation-site cluster in the bradykinin receptor (B 2 R), analogous to that present in the vasopressin receptor (V 2 R), reverses the contribution of βarr1 in ERK1/2 activation from inhibitory to promotive. An intrabody sensor suggests a conformational mechanism for this role reversal of βarr1, and molecular dynamics simulation reveals a bifurcated salt bridge between this double-phosphorylation site cluster and Lys 294 in the lariat loop of βarr1, which directs the orientation of the lariat loop. Our findings provide novel insights into the opposite roles of βarr1 in ERK1/2 activation for different GPCRs with a direct relevance to biased agonism and novel therapeutics., (© 2020 The Authors.)

A comprehensive understanding of signalling downstream of GPCRs requires a broad approach to capture novel signalling modalities in addition to established pathways. Here, using an array of sixteen validated BRET-based biosensors, we analyzed the ability of seven different β-adrenergic ligands to engage five distinct signalling pathways downstream of the β 1 -adrenergic receptor (β 1 AR). In addition to generating signalling signatures and capturing functional selectivity for the different ligands toward these pathways, we also revealed coupling to signalling pathways that have not previously been ascribed to the βAR. These include coupling to G z and G 12 pathways. The signalling cascade linking the β 1 AR to calcium mobilization was also characterized using a combination of BRET-based biosensors and CRISPR-engineered HEK 293 cells lacking the Gαs subunit or with pharmacological or genetically engineered pathway inhibitors. We show that both G s and G 12 are required for the full calcium response. Our work highlights the power of combining signal profiling with genome editing approaches to capture the full complement of GPCR signalling activities in a given cell type and to probe their underlying mechanisms.

The angiotensin II (AngII) type 1 receptor (AT1R) is a member of the G protein-coupled receptor (GPCR) family and binds β-arrestins (β-arrs), which regulate AT1R signaling and trafficking. These processes can be biased by different ligands or mutations in the AGTR1 gene. As for many GPCRs, the exact details for AT1R-β-arr interactions driven by AngII or β-arr-biased ligands remain largely unknown. Here, we used the amber-suppression technology to site-specifically introduce the unnatural amino acid (UAA) p -azido-l-phenylalanine (azF) into the intracellular loops (ICLs) and the C-tail of AT1R. Our goal was to generate competent photoreactive receptors that can be cross-linked to β-arrs in cells. We performed UV-mediated photolysis of 25 different azF-labeled AT1Rs to cross-link β-arr1 to AngII-bound receptors, enabling us to map important contact sites in the C-tail and in the ICL2 and ICL3 of the receptor. The extent of AT1R-β-arr1 cross-linking among azF-labeled receptors differed, revealing variability in β-arr's contact mode with the different AT1R domains. Moreover, the signature of ligated AT1R-β-arr complexes from a subset of azF-labeled receptors also differed between AngII and β-arr-biased ligand stimulation of receptors and between azF-labeled AT1R bearing and that lacking a bias signaling mutation. These observations further implied distinct interaction modalities of the AT1R-β-arr1 complex in biased signaling conditions. Our findings demonstrate that this photocross-linking approach is useful for understanding GPCR-β-arr complexes in different activation states and could be extended to study other protein-protein interactions in cells., (© 2019 Gagnon et al.)

The β-arrestins (β-arrs) were initially appreciated for the roles they play in the desensitization and endocytosis of G protein-coupled receptors (GPCRs). They are now also known to act as multifunctional adaptor proteins binding many non-receptor protein partners to control multiple signalling pathways. β-arrs therefore act as key regulatory hubs at the crossroads of external cell inputs and functional outputs in cellular processes ranging from gene transcription to cell growth, survival, cytoskeletal regulation, polarity, and migration. An increasing number of studies have also highlighted the scaffolding roles β-arrs play in vivo in both physiological and pathological conditions, which opens up therapeutic avenues to explore. In this introductory review chapter, we discuss the functional roles that β-arrs exert to control GPCR function, their dynamic scaffolding roles and how this impacts signal transduction events, compartmentalization of β-arrs, how β-arrs are regulated themselves, and how the combination of these events culminates in cellular regulation.

β-Arrestins are adaptors that regulate the signaling and trafficking of G protein-coupled receptors (GPCRs). Bioluminescence resonance energy transfer (BRET) is a sensitive and versatile method for real-time monitoring of protein-protein interactions and protein kinesis within live cells, such as the recruitment of β-arrestins to activated receptors at the plasma membrane (PM) and the trafficking of GPCR/β-arrestin complexes to endosomes. Trafficking of receptor/β-arrestin complexes can be assessed by BRET through tagging β-arrestins with the donor luciferase from Renilla reniformis (Rluc) and anchoring the acceptor green fluorescent protein from the same species (rGFP) in distinct cell compartments (e.g., PM or endosomes) to generate highly efficient bystander BRET (referred to as enhanced bystander BRET (EbBRET)) upon re-localization of β-arrestins to these compartments following receptor activation. Here, we outline the protocol for quantitatively monitoring β-arrestin recruitment to agonist-activated Angiotensin II type 1 receptor (AT1R) and β2-adrenergic receptor (β2AR) at the PM and the trafficking of receptor/β-arrestin complexes into endosomes using EbBRET-based biosensors.

Frizzleds (FZDs) are a group of seven transmembrane-spanning (7TM) receptors that belong to class F of the G protein-coupled receptor (GPCR) superfamily. FZDs bind WNT proteins to stimulate diverse signaling cascades involved in embryonic development, stem cell regulation, and adult tissue homeostasis. Frizzled 5 (FZD 5 ) is one of the most studied class F GPCRs that promote the functional inactivation of the β-catenin destruction complex in response to WNTs. However, whether FZDs function as prototypical GPCRs has been heavily debated and, in particular, FZD 5 has not been shown to activate heterotrimeric G proteins. Here, we show that FZD 5 exhibited a conformational change after the addition of WNT-5A, which is reminiscent of class A and class B GPCR activation. In addition, we performed several live-cell imaging and spectrometric-based approaches, such as dual-color fluorescence recovery after photobleaching (dcFRAP) and resonance energy transfer (RET)-based assays that demonstrated that FZD 5 activated Gα q and its downstream effectors upon stimulation with WNT-5A. Together, these findings suggest that FZD 5 is a 7TM receptor with a bona fide GPCR activation profile and suggest novel targets for drug discovery in WNT-FZD signaling., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

G protein-coupled receptors (GPCRs) are important therapeutic targets that exhibit functional selectivity (biased signaling), in which different ligands or receptor variants elicit distinct downstream signaling. Understanding all the signaling events and biases that contribute to both the beneficial and adverse effects of GPCR stimulation by given ligands is important for drug discovery. Here, we report the design, validation, and use of pathway-selective bioluminescence resonance energy transfer (BRET) biosensors that monitor the engagement and activation of signaling effectors downstream of G proteins, including protein kinase C (PKC), phospholipase C (PLC), p63RhoGEF, and Rho. Combined with G protein and β-arrestin BRET biosensors, our sensors enabled real-time monitoring of GPCR signaling at different levels in downstream pathways in both native and engineered cells. Profiling of the responses to 14 angiotensin II (AngII) type 1 receptor (AT1R) ligands enabled the clustering of compounds into different subfamilies of biased ligands and showed that, in addition to the previously reported functional selectivity between Gα q and β-arrestin, there are also biases among G protein subtypes. We also demonstrated that biases observed at the receptor and G protein levels propagated to downstream signaling pathways and that these biases could occur through the engagement of different G proteins to activate a common effector. We also used these tools to determine how naturally occurring AT1R variants affected signaling bias. This suite of BRET biosensors provides a useful resource for fingerprinting biased ligands and mutant receptors and for dissecting functional selectivity at various levels of GPCR signaling., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

G protein-coupled receptors (GPCRs) use diverse mechanisms to regulate the mitogen-activated protein kinases ERK1/2. β-Arrestins (βArr1/2) are ubiquitous inhibitors of G protein signaling, promoting GPCR desensitization and internalization and serving as scaffolds for ERK1/2 activation. Studies using CRISPR/Cas9 to delete βArr1/2 and G proteins have cast doubt on the role of β-arrestins in activating specific pools of ERK1/2. We compared the effects of siRNA-mediated knockdown of βArr1/2 and reconstitution with βArr1/2 in three different parental and CRISPR-derived βArr1/2 knockout HEK293 cell pairs to assess the effect of βArr1/2 deletion on ERK1/2 activation by four G s -coupled GPCRs. In all parental lines with all receptors, ERK1/2 stimulation was reduced by siRNAs specific for βArr2 or βArr1/2. In contrast, variable effects were observed with CRISPR-derived cell lines both between different lines and with activation of different receptors. For β 2 adrenergic receptors (β 2 ARs) and β 1 ARs, βArr1/2 deletion increased, decreased, or had no effect on isoproterenol-stimulated ERK1/2 activation in different CRISPR clones. ERK1/2 activation by the vasopressin V 2 and follicle-stimulating hormone receptors was reduced in these cells but was enhanced by reconstitution with βArr1/2. Loss of desensitization and receptor internalization in CRISPR βArr1/2 knockout cells caused β 2 AR-mediated stimulation of ERK1/2 to become more dependent on G proteins, which was reversed by reintroducing βArr1/2. These data suggest that βArr1/2 function as a regulatory hub, determining the balance between mechanistically different pathways that result in activation of ERK1/2, and caution against extrapolating results obtained from βArr1/2- or G protein-deleted cells to GPCR behavior in native systems., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Multiple quantitative trait loci for blood pressure (BP) are localized in humans and rodent models. Model studies have not only produced human quantitative trait loci homologues but also provided unforeseen mechanistic insights into the function modality of quantitative trait loci actions. Presently, congenic knockins, gene-specific knockout, and in vitro and in vivo function studies were used in a rat model of polygenic hypertension, DSS (Dahl salt sensitive) rats. One gene previously unknown in regulating BP was detected with 1 structural mutation(s) for each of 2 quantitative trait loci classified into 2 separate epistatic modules 1 and 3. C17QTL1 in epistatic module 2 was identified to be the gene Chrm3 encoding the M3R (muscarinic cholinergic 3 receptor), since a single function-enhancing M3R T556M conversion correlated with elevated BP. To definitively prove that the enhanced M3R function is responsible for BP changes by the DSS alleles of C17QTL1, we generated a Chrm3 gene-specific rat knockout. We observed a reduction in BP without tachycardia in both sexes, regardless of the amount of dietary salt, and an improvement in diastolic and kidney dysfunctions. All occurred in spite of a significant reduction in M3R-dependent vasodilation. The previously seen sexual dimorphism for C17QTL1 on BP disappeared in the absence of M3R. A Chrm3-coding variation increased M3R signaling, correlating with higher BP. Removing the M3R signaling led to a decrease in BP and improvements in cardiac and renal malfunctions. A novel pathogenic pathway accounted for a portion of polygenic hypertension and has implications in applying new diagnostic and therapeutic uses against hypertension and diastolic dysfunction.

The dopamine D2 receptor (D2R) is known to elicit effects through activating two major signaling pathways mediated by either G proteins (Gi/o) or β-arrestins. However, the specific role of each pathway in physiological or therapeutic activities is not known with certainty. One approach to the dissection of these pathways is through the use of drugs that can selectively modulate one pathway vs. the other through a mechanism known as functional selectivity or biased signaling. Our laboratory has previously described a G protein signaling-biased agonist, MLS1547, for the D2R using a variety of in vitro functional assays. To further evaluate the biased signaling activity of this compound, we investigated its ability to promote D2R internalization, a process known to be mediated by β-arrestin. Using multiple cellular systems and techniques, we found that MLS1547 promotes little D2R internalization, which is consistent with its inability to recruit β-arrestin. Importantly, we validated these results in primary striatal neurons where the D2R is most highly expressed suggesting that MLS1547 will exhibit biased signaling activity in vivo . In an effort to optimize and further explore structure-activity relationships (SAR) for this scaffold, we conducted an iterative chemistry campaign to synthesize and characterize novel analogs of MLS1547. The resulting analysis confirmed previously described SAR requirements for G protein-biased agonist activity and, importantly, elucidated new structural features that are critical for agonist efficacy and signaling bias of the MLS1547 scaffold. One of the most important determinants for G protein-biased signaling is the interaction of a hydrophobic moiety of the compound with a defined pocket formed by residues within transmembrane five and extracellular loop two of the D2R. These results shed new light on the mechanism of biased signaling of the D2R and may lead to improved functionally-selective molecules.

In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, β-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of β-arrestin recruitment to the receptor and β-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 β-arrestin and the β2-adaptin subunit of the clathrin adaptor protein AP2 without interfering with the formation of receptor/β-arrestin complexes. This selective β-arrestin/β2-adaptin inhibitor (Barbadin) blocks agonist-promoted endocytosis of the prototypical β2-adrenergic (β2AR), V2-vasopressin (V2R) and angiotensin-II type-1 (AT1R) receptors, but does not affect β-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 β2AR, supporting the concept of β-arrestin/AP2-dependent signalling for both G protein-dependent and -independent pathways.

Lymphangioleiomyomatosis (LAM) is a destructive lung disease that can arise sporadically or in adults suffering from the tumor syndrome tuberous sclerosis complex (TSC). Microscopic tumors ('LAM nodules') in the lung interstitium arise from lymphatic invasion and metastasis. These consist of smooth muscle-like cells (LAM cells) that exhibit markers of neural crest differentiation and loss of the tumor suppressor protein 'tuberous sclerosis complex-2' (TSC2). Consistent with a neural phenotype, expression of the neuropeptide urotensin-II and its receptor was detected in LAM nodules. We hypothesized that loss of TSC2 sensitizes cells to the oncogenic effects of urotensin-II. TSC2-deficient Eker rat uterine leiomyoma ELT3 cells were stably transfected with empty vector or plasmid for the expression of TSC2. Urotensin-II increased cell viability and proliferation in TSC2-deficient cells, but not in TSC2-reconstituted cells. When exposed to urotensin-II, TSC2-deficient cells exhibited greater migration, anchorage-independent cell growth, and matrix invasion. The effects of urotensin-II on TSC2-deficient cells were blocked by the urotensin receptor antagonist SB657510, and accompanied by activation of Erk mitogen-activated protein kinase and focal adhesion kinase. Urotensin-II-induced proliferation and migration were reproduced in TSC2-deficient human angiomyolipoma cells, but not in those stably expressing TSC2. In a mouse xenograft model, SB657510 blocked the growth of established ELT3 tumors, reduced the number of circulating tumor cells, and attenuated the production of VEGF-D, a clinical biomarker of LAM. Urotensin receptor antagonists may be selective therapeutic agents for the treatment of LAM or other neural crest-derived neoplasms featuring loss of TSC2 or increased expression of the urotensin receptor., Competing Interests: None of the authors declare financial interests or relationships that would represent a conflict of interest with respect to the subject matter of this manuscript.

Endocytosis and intracellular trafficking of receptors are pivotal to maintain physiological functions and drug action; however, robust quantitative approaches are lacking to study such processes in live cells. Here we present new bioluminescence resonance energy transfer (BRET) sensors to quantitatively monitor G protein-coupled receptors (GPCRs) and β-arrestin trafficking. These sensors are based on bystander BRET and use the naturally interacting chromophores luciferase (RLuc) and green fluorescent protein (rGFP) from Renilla. The versatility and robustness of this approach are exemplified by anchoring rGFP at the plasma membrane or in endosomes to generate high dynamic spectrometric BRET signals on ligand-promoted recruitment or sequestration of RLuc-tagged proteins to, or from, specific cell compartments, as well as sensitive subcellular BRET imaging for protein translocation visualization. These sensors are scalable to high-throughput formats and allow quantitative pharmacological studies of GPCR trafficking in real time, in live cells, revealing ligand-dependent biased trafficking of receptor/β-arrestin complexes.

G protein-coupled receptors (GPCRs) are known to initiate a plethora of signaling pathways in vitro. However, it is unclear which of these pathways are engaged to mediate physiological responses. Here, we examine the distinct roles of Gq/11-dependent signaling and receptor phosphorylation-dependent signaling in bronchial airway contraction and lung function regulated through the M3-muscarinic acetylcholine receptor (M3-mAChR). By using a genetically engineered mouse expressing a G protein-biased M3-mAChR mutant, we reveal the first evidence, to our knowledge, of a role for M3-mAChR phosphorylation in bronchial smooth muscle contraction in health and in a disease state with relevance to human asthma. Furthermore, this mouse model can be used to distinguish the physiological responses that are regulated by M3-mAChR phosphorylation (which include control of lung function) from those responses that are downstream of G protein signaling. In this way, we present an approach by which to predict the physiological/therapeutic outcome of M3-mAChR-biased ligands with important implications for drug discovery.

Arrestins are cytosolic proteins that regulate G-protein-coupled receptor (GPCR) desensitization, internalization, trafficking and signalling. Arrestin recruitment uncouples GPCRs from heterotrimeric G proteins, and targets the proteins for internalization via clathrin-coated pits. Arrestins also function as ligand-regulated scaffolds that recruit multiple non-G-protein effectors into GPCR-based 'signalsomes'. Although the dominant function(s) of arrestins vary between receptors, the mechanism whereby different GPCRs specify these divergent functions is unclear. Using a panel of intramolecular fluorescein arsenical hairpin (FlAsH) bioluminescence resonance energy transfer (BRET) reporters to monitor conformational changes in β-arrestin2, here we show that GPCRs impose distinctive arrestin 'conformational signatures' that reflect the stability of the receptor-arrestin complex and role of β-arrestin2 in activating or dampening downstream signalling events. The predictive value of these signatures extends to structurally distinct ligands activating the same GPCR, such that the innate properties of the ligand are reflected as changes in β-arrestin2 conformation. Our findings demonstrate that information about ligand-receptor conformation is encoded within the population average β-arrestin2 conformation, and provide insight into how different GPCRs can use a common effector for different purposes. This approach may have application in the characterization and development of functionally selective GPCR ligands and in identifying factors that dictate arrestin conformation and function.

Angiotensin II (Ang II) is a vasopressive hormone but is also a potent activator of cellular migration. We have previously shown that it can promote the activation of the GTPase ARF6 in a heterologous overexpressing system. The molecular mechanisms by which receptors control the activation of this small G protein remain, however, largely unknown. Furthermore, how ARF6 coordinates the activation of complex cellular responses needs to be further elucidated. In this study, we demonstrate that Ang II receptors engage β-arrestin, but not Gq, to mediate ARF6 activation in HEK 293 cells. To further confirm the key role of β-arrestin proteins, we overexpressed β-arrestin2-(1-320), a dominant negative mutant known to block receptor endocytosis. We show that expression of this truncated construct does not support the activation of the GTPase nor cell migration. Interestingly, β-arrestin2 can interact with the ARF guanine nucleotide exchange factor ARNO, although the C-terminally lacking mutant does not. We finally examined whether receptor endocytosis controlled ARF6 activation and cell migration. Although the clathrin inhibitor PitStop2 did not impact the ability of Ang II to activate ARF6, cell migration was markedly impaired. To further show that ARF activation regulates key signaling events leading to migration, we also examined MAPK activation. We demonstrate that this signaling axis is relevant in smooth muscle cells of the vasculature. Altogether, our findings show for the first time that Ang II receptor signaling to β-arrestin regulates ARF6 activation. These proteins together control receptor endocytosis and ultimately cell migration., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

There has been a growing appreciation that G protein-coupled receptor (GPCR) functional selectivity (viz. biased signaling), in particular between G protein- and β-arrestin-dependent signaling, can be achieved with specific ligands, and that such directed signaling represents a promising avenue for improving drug efficacy and therapy. Thus, for any given GPCRs it is important to define means to pharmacologically characterize and classify drugs for their propensity to bias signaling. Here we describe an experimental protocol and step-by-step approach to assess functional selectivity between Gαq and β-arrestin-dependent responses using the prototypical angiotensin II (AngII) type 1 receptor (AT1R) expressed in HEK 293 cells. The protocol describes the expression of Bioluminescence Resonance Energy Transfer (BRET) sensors for either Gαq or β-arrestin with AT1R, and the use of the operational model of pharmacological agonism to quantify ligand bias. Such methods are equally applicable to other GPCRs and their downstream signaling effectors., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Despite the discovery of heterotrimeric αβγ G proteins ∼25 years ago, their selective perturbation by cell-permeable inhibitors remains a fundamental challenge. Here we report that the plant-derived depsipeptide FR900359 (FR) is ideally suited to this task. Using a multifaceted approach we systematically characterize FR as a selective inhibitor of Gq/11/14 over all other mammalian Gα isoforms and elaborate its molecular mechanism of action. We also use FR to investigate whether inhibition of Gq proteins is an effective post-receptor strategy to target oncogenic signalling, using melanoma as a model system. FR suppresses many of the hallmark features that are central to the malignancy of melanoma cells, thereby providing new opportunities for therapeutic intervention. Just as pertussis toxin is used extensively to probe and inhibit the signalling of Gi/o proteins, we anticipate that FR will at least be its equivalent for investigating the biological relevance of Gq.

On pursuing molecules that delay labour, so-called tocolytics, the prostaglandin F2α receptor (FP) was targeted, because of its role in the stimulation of uterine contractions leading to birth and preterm birth. Previously, both the indolizidinone PDC-113.824 (5) and the aza-glycinyl-proline analog 6 were shown to delay labour in mice by modulating the FP function, likely by an allosteric mechanism, which features biased signalling. The crystal structure and computational analyses of the indolizidin-2-one amino acid and aza-glycinyl-proline components of 5 and 6 in model peptides have shown them to adopt a geometry that mimics ideal type I and II'β-turns. To elucidate the precise turn geometry for receptor recognition, analogs 1-4 have now been synthesized: macrocycle and pyrroloazepinone mimics 1 and 2 to mimic type I, and glycinyl-proline and d-alaninyl-proline analogs 3 and 4 to favour type II'β-turn geometry. Notably, transannular cyclization of peptide macrocycle 13 has provided diastereoselectively pyrroloazepinone 15 by a novel route that provides effective access to mimics 1 and 2 by way of a common intermediate. Among the four analogs, none exhibited efficacy nor potency on par with 5 and 6; however, d-alaninyl-proline analog 4 proved superior to the other analogs in reducing PGF2α-induced myometrial contractions and inhibiting FP modulation of cell ruffling, a response dependent on the Gα12/RhoA/ROCK signaling pathway. Furthermore Gly-Pro analog 3 potentiated the effect of PGF2α on Gαq mediated ERK1/2 activation. Evidence that 4 adopted turn geometry was obtained by conformational analysis using NMR spectroscopy to characterize respectively the influence of solvent and temperature on the chemical shifts of the amide NH protons. Although mimicry of the type II' geometry by 3, 4, 5 and 6 may favour activity, distortion from ideal geometry by the indolizidinone and aza-glycinyl residues of the latter appears to enhance their biological effects.

The angiotensin II type I (AT1R) and the prostaglandin F2α (PGF2α) F prostanoid (FP) receptors are both potent regulators of blood pressure. Physiological interplay between AT1R and FP has been described. Abdominal aortic ring contraction experiments revealed that PGF2α-dependent activation of FP potentiated angiotensin II-induced contraction, whereas FP antagonists had the opposite effect. Similarly, PGF2α-mediated vasoconstriction was symmetrically regulated by co-treatment with AT1R agonist and antagonist. The underlying canonical Gαq signaling via production of inositol phosphates mediated by each receptor was also regulated by antagonists for the other receptor. However, binding to their respective agonists, regulation of receptor-mediated MAPK activation and vascular smooth muscle cell growth were differentially or asymmetrically regulated depending on how each of the two receptors were occupied by either agonist or antagonist. Physical interactions between these receptors have never been reported, and here we show that AT1R and FP form heterodimeric complexes in both HEK 293 and vascular smooth muscle cells. These findings imply that formation of the AT1R/FP dimer creates a novel allosteric signaling unit that shows symmetrical and asymmetrical signaling behavior, depending on the outcome measured. AT1R/FP dimers may thus be important in the regulation of blood pressure., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

β-Arrestins are signaling adaptors that bind to agonist-occupied G protein-coupled receptors (GPCRs) and target them for endocytosis; however, the mechanisms regulating receptor/β-arrestin complexes and trafficking in endosomes, remain ill defined. Here we show, in live cells, differential dynamic regulation of endosomal bradykinin B2 receptor (B2R) complexes with either β-arrestin-1 or -2. We find a novel role for MAPK in the B2R/β-arrestin-2 complex formation, receptor trafficking and signaling mediated by an ERK1/2 regulatory motif in the hinge domain of the rat β-arrestin-2 (PET(178)P), but not rat β-arrestin-1 (PER(177)P). While the ERK1/2 regulatory motif is conserved between rat and mouse β-arrestin-2, it is surprisingly not conserved in human β-arrestin-2 (PEK(178)P). However, mutation of lysine 178 to threonine is sufficient to confer MAPK sensitivity to the human β-arrestin-2. Furthermore, substitution for a phosphomimetic residue in both the rat and the human β-arrestin-2 (T/K178D) significantly stabilizes B2R/β-arrestin complexes in endosomes, delays receptor recycling to the plasma membrane and maintains intracellular MAPK signaling. Similarly, the endosomal trafficking of β2-adrenergic, angiotensin II type 1 and vasopressin V2 receptors was altered by the β-arrestin-2 T178D mutant. Our findings unveil a novel subtype specific mode of MAPK-dependent regulation of β-arrestins in intracellular trafficking and signaling of GPCRs, and suggest differential endosomal receptor/β-arrestin-2 signaling roles among species., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that participate in many aspects of the endocrine function and are important targets for drug development. They transduce signals mainly, but not exclusively, via hetero-trimeric G proteins, leading to a diversity of intracellular signaling cascades. Ligands binding at the hormone orthosteric sites of receptors have been classified as agonists, antagonists, and/or inverse agonists based on their ability to mainly modulate G protein signaling. Accumulating evidence also indicates that such ligands, alone or in combination with other ones such as those acting outside the orthosteric hormone binding sites (e.g., allosteric modulators), have the ability to selectively engage subsets of signaling responses as compared to the natural endogenous ligands. Such modes of functioning have been variously referred to as "functional selectivity" or "ligand-biased signaling." In this review, we provide an overview of the current knowledge regarding GPCR-biased signaling and their functional regulation with a focus on the evolving concept that receptor domains can also be targeted to allosterically bias signaling, and discuss the usefulness of such modes of regulation for the design of more efficient therapeutics.

Allergic asthma is a heterogeneous disease with no curative therapies. T cells infiltrate the airway smooth muscle (ASM) layer and may be implicated in airway remodeling and the increase of ASM mass, a cardinal feature of asthma. The mechanism by which CD4(+) T cells drive airway remodeling remains unknown. This study sought to determine the T cell-mediated mechanism of ASM cell proliferation. We hypothesized that CD4(+) T cells adhere to ASM cells via CD44, and induce ASM cell proliferation through the activation of the epidermal growth factor receptor (EGFR). A coculture model showed that the contact of antigen-stimulated CD4(+) T cells with ASM cells induced high levels of EGFR ligand expression in CD4(+) T cells and the activation of matrix metalloproteinase (MMP)-9, required for the shedding of EGFR ligands. The inhibition of EGFR and MMP-9 prevented the increase of ASM cell proliferation after coculture. The hyaluronan receptor CD44 is the dominant mediator of the tight adherence of T cells to ASM and is colocalized with MMP-9 on the cell surface. Moreover, the neutralization of CD44 prevents ASM cell hyperplasia. These data provide a novel mechanism by which antigen-stimulated CD4(+) T cells induce the remodeling indicative of a direct trophic role for CD4(+) T cells.

Unlabelled: Dimers of GPCRs have held the imagination of researchers for almost 20 years. However, only recently has their value as potentially novel drug targets been increased significantly, and primarily, in the context of GPCR heterodimers. The view of receptor heterodimers as allosteric machines has transformed the way we understand structural and functional asymmetries inherent in their organization. These asymmetries alter both signalling output and how they might be targeted pharmacologically. The paper in this issue of BJP by Siddiquee and colleagues () highlights our growing understanding of such asymmetries and their implications. They show that heterodimers of the angiotensin II AT1 receptor and the apelin receptor recognize and respond to their respective ligands in distinct ways from the parent receptors expressed alone. Further, they demonstrate asymmetric allosteric effects in the context of the heterodimer that may have significant implications for our understanding of such receptor complexes., Linked Article: This article is a commentary on the research paper by Siddiquee et al., pp. 1104-1117 of this issue. To view this paper visit http://dx.doi.org/10.1111/j.1476-5381.2012.02192.x., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

G protein-coupled receptors (GPCRs) represent one of the largest families of cell surface receptors as key targets for pharmacological manipulation. G proteins have long been recognized as allosteric modulators of GPCR ligand binding. More recently, small molecule allosteric modulators have now been widely characterized for a number of GPCRs, and some are now used clinically. Many studies have also underscored the importance of GPCR dimerization or higher-order oligomerization in the control of the physiological responses they modulate. Thus, allosterism can also, between monomer equivalents in the context of a dimer, oligomer, or receptor mosaic, influence signaling pathways downstream. It therefore becomes essential to characterize both small molecule allosteric ligands and allosteric interactions between receptors modulated by canonical orthosteric ligands, in a pathway-specific manner. Here, we describe a simple, radioligand-binding method, which is designed to probe for allosteric modulation mediated by any GPCR interactor, from small molecules to interacting proteins. It can also detect allosteric asymmetries within a GPCR heterodimer, via orthosteric or allosteric ligands. This assay measures time-dependent ligand occupancy of radiolabeled orthosteric or (with adaptations) allosteric ligands as modulated by either small molecules or receptor dimer partners bound or unbound with their own ligands., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The great versatility of G protein-coupled receptors (GPCRs), in terms of both their ability to bind different types of ligands and initiate a large number of distinct cellular signaling events, remains incompletely understood. In recent years, the classical view of the nature and consequences of ligand binding to GPCRs has dramatically changed. The notion of functional selectivity, achieved through both biased ligands and allosteric modulators, has brought substantial new insight into our comprehension of the pluridimensionality of signaling achieved by GPCRs. Moreover, receptor heterodimerization adds another important dimension to the diversity of cellular responses controlled by GPCRs. Here, we review these considerations and discuss how they will impact the design of improved therapeutics.

The G protein-coupled prostaglandin F2α (PGF2α) receptor [F prostanoid (FP) receptor] has been implicated in many physiological events including cardiovascular, respiratory, immune, reproductive, and endocrine responses. Binding of PGF2α to FP receptor elicits inositol production and protein kinase C-dependent MAPK activation through Gα(q) coupling. Here we report that AL-8810, previously characterized as an orthosteric antagonist of PGF2α-dependent, Gα(q)-mediated signaling, potently activates ERK1/2 in a protein kinase C-independent manner. Rather, AL-8810 promoted ERK1/2 activation via an epidermal growth factor receptor transactivation mechanism in both human embryonic kidney 293 cells and in the MG-63 osteoblast-like cells, which express endogenous FP receptors. Neither AL-8810- nor PGF2α-mediated stimulation of FP receptor promoted association with β-arrestins, suggesting that MAPK activation induced by these ligands is independent of β-arrestin's signaling scaffold functions. Interestingly, the spatiotemporal activation of ERK1/2 promoted by AL-8810 and PGF2α showed almost completely opposite responses in the nucleus and the cytosol. Finally, using [(3)H]thymidine incorporation, we noted differential regulation of PGF2α- and AL-8810-induced cell proliferation in MG-63 cells. This study reveals, for the first time, the signaling biased nature of FP receptor orthosteric ligands toward MAPK signaling. Our findings on the specific patterns of ERK1/2 activation promoted by FP receptor ligands may help dissect the distinct roles of MAPK in FP receptor-dependent physiological responses.

The angiotensin type 1 receptor (AT1R) and its octapeptide ligand, angiotensin II (AngII), engage multiple downstream signaling pathways, including those mediated by heterotrimeric guanosine triphosphate-binding proteins (G proteins) and those mediated by β-arrestin. Here, we examined AT1R-mediated Gα(q) and β-arrestin signaling with multiple AngII analogs bearing substitutions at position 8, which is critical for binding to the AT1R and its activation of G proteins. Using assays that discriminated between ligand-promoted recruitment of β-arrestin to the AT1R and its resulting conformational rearrangement, we extend the concept of biased signaling to include the analog's propensity to differentially promote conformational changes in β-arrestin, two responses that were differentially affected by distinct G protein-coupled receptor kinases. The efficacy of AngII analogs in activating extracellular signal-regulated kinases 1 and 2 correlated with the stability of the complexes between β-arrestin and AT1R in endosomes, rather than with the extent of β-arrestin recruitment to the receptor. In vascular smooth muscle cells, the ligand-induced conformational changes in β-arrestin correlated with whether the ligand promoted β-arrestin-dependent migration or proliferation. Our data indicate that biased signaling not only occurs between G protein- and β-arrestin-mediated pathways but also occurred at the level of the AT1R and β-arrestin, such that different AngII analogs selectively engaged distinct β-arrestin conformations, which led to specific signaling events and cell responses.

The Gq-coupled oxytocin receptor (OTR) and the Gs-coupled β(2)-adrenergic receptor (β(2)AR) are both expressed in myometrial cells and mediate uterine contraction and relaxation, respectively. The two receptors represent important pharmacological targets as OTR antagonists and β(2)AR agonists are used to control pre-term uterine contractions. Despite their physiologically antagonistic effects, both receptors activate the MAP kinases ERK1/2, which has been implicated in uterine contraction and the onset of labor. To determine the signalling pathways involved in mediating the ERK1/2 response, we assessed the effect of blockers of specific G protein-associated pathways. In human myometrial hTERT-C3 cells, inhibition of Gαi as well as inhibition of the Gαq/PKC pathway led to a reduction of both OTR- and β(2)AR-mediated ERK1/2 activation. The involvement of Gαq/PKC in β(2)AR-mediated ERK1/2 induction was unexpected. To test whether the emergence of this novel signalling mechanism was dependent on OTR expression in the same cell, we conducted experiments in HEK 293 cells that were transfected with the β(2)AR alone or co-transfected with the OTR. Using this approach, we found that β(2)AR-mediated ERK1/2 responses became sensitive to PKC inhibition only in cells co-transfected with the OTR. Inhibitor studies indicated the involvement of an atypical PKC isoform in this process. We confirmed the specific involvement of PKCζ in this pathway by assessing PKCζ translocation to the cell membrane. Consistent with our inhibitor studies, we found that β(2)AR-mediated PKCζ translocation was dependent on co-expression of OTR. The present demonstration of a novel β(2)AR-coupled signalling pathway that is dependent on OTR co-expression is suggestive of a molecular interaction between the two receptors., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The oxytocin receptor (OTR) and the β(2)-adrenergic receptor (β(2)AR) are key regulators of uterine contraction. These two receptors are targets of tocolytic agents used to inhibit pre-term labor. Our recent study on the nature of OTR- and β(2)AR-mediated ERK1/2 activation in human hTERT-C3 myometrial cells suggested the presence of an OTR/β(2)AR hetero-oligomeric complex (see companion article). The goal of this study was to investigate potential allosteric interactions between OTR and β(2)AR and establish the nature of the interactions between these receptors in myometrial cells. We found that OTR-mediated ERK1/2 activation was attenuated significantly when cells were pretreated with the β(2)AR agonist isoproterenol or two antagonists, propranolol or timolol. In contrast, pretreatment of cells with a third β(2)AR antagonist, atenolol resulted in an increase in OTR-mediated ERK1/2 activation. Similarly, β(2)AR-mediated ERK1/2 activation was strongly attenuated by pretreatment with the OTR antagonists, atosiban and OTA. Physical interactions between OTR and β(2)AR were demonstrated using co-immunoprecipitation, bioluminescence resonance energy transfer (BRET) and protein-fragment complementation (PCA) assays in HEK 293 cells, the latter experiments indicating the interactions between the two receptors were direct. Our analyses suggest physical interactions between OTR and β(2)AR in the context of a new heterodimer pair lie at the heart of the allosteric effects., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The prostaglandin-F2α (PGF2α) receptor (FP) was targeted to develop tocolytic agents for inhibiting preterm labor. Azabicycloalkane and azapeptide mimics 2-10 were synthesized based on the (3S,6S,9S)-indolizidin-2-one amino acid analogue PDC113.824 (1), which was shown to modulate FP by a biased allosteric mechanism, involving both Gαq- and Gα12-mediated signaling pathways, and exhibited significant tocolytic activity delaying preterm labor in a mouse model ( Goupil ; et al. J. Biol. Chem. 2010 , 285 , 25624 - 25636 ). Although changes in azabicycloalkane stereochemistry and ring size caused loss of activity, replacement of the indolizidin-2-one amino acid with azaGly-Pro and azaPhe-Pro gave azapeptides 6 and 8, which reduced PGF2α-induced myometrial contractions, potentiated the effect of PGF2α on Gαq-mediated ERK1/2 activation, and inhibited FP modulation of cell ruffling, a response dependent on the Gα12/RhoA/ROCK signaling pathway. Revealing complementarities of azabicycloalkane and azapeptide mimics, novel probes, and efficient tocolytic agents were made to study allosteric modulation of the FP receptor.

The type II transmembrane serine protease TMPRSS6 (also known as matriptase-2) controls iron homeostasis through its negative regulation of expression of hepcidin, a key hormone involved in iron metabolism. Upstream of the hepcidin-regulated signaling pathway, TMPRSS6 cleaves its target substrate hemojuvelin (HJV) at the plasma membrane, but the dynamics of the cell-surface expression of the protease have not been addressed. Here, we report that TMPRSS6 undergoes constitutive internalization in transfected HEK293 cells and in two human hepatic cell lines, HepG2 and primary hepatocytes, both of which express TMPRSS6 endogenously. Cell surface-labeled TMPRSS6 was internalized and was detected in clathrin- and AP-2-positive vesicles via a dynamin-dependent pathway. The endocytosed TMPRSS6 next transited in early endosomes and then to lysosomes. Internalization of TMPRSS6 is dependent on specific residues within its N-terminal cytoplasmic domain, as site-directed mutagenesis of these residues abrogated internalization and maintained the enzyme at the cell surface. Cells coexpressing these mutants and HJV produced significantly decreased levels of hepcidin compared with wild-type TMPRSS6 due to the sustained cleavage of HJV at the cell surface by TMPRSS6 mutants. Our results underscore for the first time the importance of TMPRSS6 trafficking at the plasma membrane in the regulation of hepcidin expression, an event that is essential for iron homeostasis.

G protein-coupled receptors (GPCRs) can engage multiple pathways to activate ERK1/2 via both G proteins and/or ßarrestin. Receptor recruitment of ßarrestin is also important for GPCR desensitization, internalization and resensitization. Modulation of the receptor/ßarrestin interaction through modification of either component would presumably alter the output generated by receptor activation. Here we examined how ßarrestins regulate bradykinin (BK) B2 receptor (B2R) signalling and desensitization by either truncating ßarrestin1 or ßarrestin2 or by alanine substitution of a serine/threonine cluster in the C-terminal tail of B2R (B2R-4A), conditions which all affect the avidity of the B2R/ßarrestin complex. We first demonstrate that BK-mediated ERK1/2 activation is biphasic containing an early peak (between 2-5min) followed by sustained activation for at least 60min. The early but not the sustained phase was predictably affected by inhibition of either Gαq/11 or Gαi/o, whereas loss of ßarrestin2 but not ßarrestin1 resulted in diminished prolonged ERK1/2 activation. ßarrestin2's role was further examined using a truncation mutant with augmented avidity for the agonist-occupied receptor, revealing an increase in both immediate and extended ERK1/2 signalling. We also show that ERK1/2 is recruited to the B2R/ßarrestin complex on endosomes as well as the plasma membrane. Moreover, we investigated ßarrestin's role using the B2R-4A, which is deficient in ßarrestin binding and does not internalize. We show that ERK1/2 signalling downstream of the receptor is entirely G protein-dependent and receptor-mediated intracellular calcium mobilization studies revealed a lack of desensitization. Functionally, the lack of desensitization resulted in increased cell growth and migration compared to the wild-type receptor, which was sensitive to MEK inhibition. These results highlight ßarrestin's crucial role in the maintenance of proper B2R signalling., (Copyright © 2010 Elsevier Inc. All rights reserved.)

L-type prostaglandin synthase (L-PGDS) produces PGD(2), a lipid mediator involved in neuromodulation and inflammation. Here, we show that L-PGDS and arrestin-3 (Arr3) interact directly and can be co-immunoprecipitated endogenously from MG-63 osteoblasts. Perinuclear L-PGDS/Arr3 co-localization is observed in PGD(2)-producing MG-63 cells and is induced by the addition of the L-PGDS substrate or co-expression of COX-2 in HEK293 cells. Inhibition of L-PGDS activity in MG-63 cells triggers redistribution of Arr3 and L-PGDS to the cytoplasm. Perinuclear localization of L-PGDS is detected in wild-type mouse embryonic fibroblasts (MEFs) but is more diffused in MEFs-arr-2(-/-)-arr-3(-/-). Arrestin-3 promotes PGD(2) production by L-PGDS in vitro. IL-1β-induced PGD(2) production is significantly lower in MEFs-arr-2(-/-)-arr-3(-/-) than in wild-type MEFs but can be rescued by expressing Arr2 or Arr3. A peptide corresponding to amino acids 86-100 of arrestin-3 derived from its L-PGDS binding domain stimulates L-PGDS-mediated PGD(2) production in vitro and in MG-63 cells. We report the first characterization of an interactor/modulator of a PGD(2) synthase and the identification of a new function for arrestin, which may open new opportunities for improving therapies for the treatment of inflammatory diseases.

β-arrestins, through their scaffolding functions, are key regulators of G protein-coupled receptor (GPCR) signaling and intracellular trafficking. However, little is known about the dynamics of β-arrestin/receptor interactions and how these complexes, and complexes with other regulatory proteins, are controlled in cells. Here, we use yellow fluorescent protein (YFP)-tagged β-arrestin 2 and a fluorescence recovery after photobleaching (FRAP) imaging approach to probe the real-time interaction of β-arrestin with a GPCR, the bradykinin type 2 receptor (B2R). We provide a detailed protocol to assess the avidity of β-arrestin2-YFP for B2R within endosomes in HEK293 cells. β-arrestin2-YFP associated with internalized receptors is photobleached with intense light, and fluorescence recovery due to the entry of nonbleached β-arrestin2-YFP is monitored over time as a measure of the rate exchange of β-arrestin2-YFP within the endosome. This approach can be extended to other GPCR/β-arrestin complexes and their putative regulators to provide information about the kinetics of similar protein-protein interactions in cells. Moreover, these techniques should provide insight into the role of β-arrestins in the intracellular trafficking and signaling of GPCRs.