G Protein-coupled Receptors Activation Research Articles

Constitutive internalisation of EP2 differentially regulates G protein signalling.

The prostanoid G protein-coupled receptor (GPCR) EP2 is widely expressed and implicated in endometriosis, osteoporosis, obesity, pre-term labour, and cancer. Internalisation and intracellular trafficking are critical for shaping GPCR activity, yet little is known regarding spatial programming of EP2 signalling and whether this can be exploited pharmacologically. Using three EP2-selective ligands that favour activation of different EP2 pathways, we show that EP2 undergoes limited agonist-driven internalisation but is constitutively internalised via dynamin-dependent, β-arrestin-independent pathways. EP2 was constitutively trafficked to early and very early endosomes (VEE) which was not altered by ligand activation. APPL1, a key adaptor and regulatory protein of the VEE, did not impact EP2 agonist-mediated cAMP. Internalisation was required for ~70% of the acute butaprost- and AH13205-mediated cAMP signalling, yet PGN9856i, a Gαs biased agonist, was less dependent on receptor internalisation for its cAMP signalling, particularly in human term pregnant myometrial cells that endogenously express EP2. Inhibition of EP2 internalisation partially reduced calcium signalling activated by butaprost or AH13205 and had no effect on PGE2 secretion. This indicates an agonist-dependent differential spatial requirement for Gαs and Gαq/11 signalling and a role for plasma membrane initiated Gαq/11-Ca2+-mediated PGE2 secretion. These findings reveal a key role for EP2 constitutive internalisation in its signalling and potential spatial bias in mediating its downstream functions. This in turn could highlight important considerations for future selective targeting of EP2 signalling pathways.

Mechanistic insights into sodium ion-mediated ligand binding affinity and modulation of 5-HT2B GPCR activity: implications for drug discovery and development

Purpose The G-protein coupled receptor (GPCR) family, implicated in neurological disorders and drug targets, includes the sensitive serotonin receptor subtype, 5-HT2B. The influence of sodium ions on ligand binding at the receptor’s allosteric region is being increasingly studied for its impact on receptor structure. Methods High-throughput virtual screening of three libraries, specifically the Asinex-GPCR library, which contains 8,532 compounds and FDA-approved (2466 compounds) and investigational compounds (2731)) against the modeled receptor [4IB4-5HT2BRM] using the standard agonist/antagonist (Ergotamine/Methysergide), as previously selected from our studies based on ADMET profiling, and further on basis of binding free energy a single compound – dihydroergotamine is chosen. Results This compound displayed strong interactions with the conserved active site. Ions influence ligand binding, with stronger interactions (3-H-bonds and 1-π-bond around 3.35 Å) observed when an agonist and ions are present. Ions entry is guided by conserved motifs in helices III, IV, and VII, which regulate the receptor. Dihydroergotamine, the selected drug, showed binding variance based on ions presence/absence, affecting amino acid residues in these motifs. DCCM and PCA confirmed the stabilization of ligands, with a greater correlation (∼46.6%-PC1) observed with ions. Dihydroergotamine-modified interaction sites within the receptor necessary for activation, serving as a potential 5HT2BRM agonist. RDF analysis showed the sodium ions density around the active site during dihydroergotamine binding. Conclusion Our study provides insights into sodium ion mobility’s role in controlling ligand binding affinity in 5HT2BR, offering therapeutic development insights.

Abstract 2760: Novel GPCR recombinant monoclonal antibodies for cancer biology research

Abstract Recombinant technology is becoming the preferred production mechanism for creation of antibody reagents used in academic and clinical cancer biology research. Recombinant antibodies (rAbs) are superior to standard polyclonal and hybridoma-generated monoclonal reagents as they offer reproducible performance and consistent, scalable supply. A major focus of antibody manufacturers is to leverage rAb production strategies to generate antibodies against challenging targets for which there are few, if any, reliable antibodies. G-protein coupled receptors (GPCRs) are encoded by more than 820 human genes and represent the largest membrane protein superfamily in the genome. These seven-pass transmembrane domain receptors are involved in a multitude of physiological and pathophysiological processes related to various chronic diseases, including an array of malignancies. Perhaps 30-40% of all therapeutics are directed against GPCR activity, and ~350 of the non-olfactory GPCRs are thought to be potential candidates for drug development. However, for a slew of technical reasons, the development and validation of high-quality antibodies against GPCRs has proven difficult. In an effort to facilitate further research into GPCRs, GeneTex is combining its recombinant antibody production platform with enhanced validation techniques to make specific monoclonal antibodies against GPCRs with direct relevance to cancer and other diseases. GeneTex’s rAb production protocol employs a multi-parameter fluorescence-activated single cell sorting (FACS)-based methodology to identify and select antigen-specific IgG+ memory B cells from an immunized rabbit (Starkie et al., 2016). The heavy and light chain variable region genes from single cells are amplified, cloned, and co-expressed in mammalian cells to generate a monoclonal IgG. Application-specific testing is performed in conjunction with knockout/knockdown (KO/KD), differential expression comparison in cells and tissues, cell fraction enrichment (i.e., membrane extracts versus whole cell extracts), and other methodologies. Antibodies directed against extracellular GPCR domains are validated for specificity using GPCR arrays (CDI Labs, Baltimore, MD). This workflow has resulted in the production of specific antibodies for a number of GPCRs studied by cancer biologists. These include novel recombinant monoclonal antibodies to the dopamine D2 receptor (DRD2), retinoic acid-induced protein 3 (RAI3), CXC motif chemokine receptor 2 (CXCR2), CXCR4, and CXCR7. All of these antibodies were validated for at least two applications, while the specificity of three (i.e., RAI3, CXCR4, and CXCR7) is supported by KO/KD data. GPCR array data argues for the specificity of the other two antibodies (i.e., DRD2 and CXCR2). This same production/validation approach will be applied as GeneTex targets the remaining GPCRs. Citation Format: Alexander Ball, Yung Lin Hsieh, Chun Kai Huang, Sega Huang, Shang Ru Wu, Yue Yun Chang, Jiming Liu, Po Shin Peng, Chia Yi Lin. Novel GPCR recombinant monoclonal antibodies for cancer biology research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2760.

Abstract 671: G-protein coupled receptor (GPCR) screening library identifies sertindole, an antipsychotic, as potential therapeutic option to target GNAQ mutation Q209L in uveal melanoma

Abstract Background: Uveal melanoma is a rare but aggressive intraocular malignancy with limited therapeutic options. A hallmark genetic alteration associated with uveal melanoma is the mutation of the GNAQ gene, particularly the Q209L substitution in the Gαq protein, which contributes to oncogenic signaling and tumor progression. The GNAQ gene encodes for a Gαq protein that plays a critical role in signal transduction pathways involving G protein-coupled receptors (GPCRs). The Q209L substitution results in a gain-of-function mutation, leading to the chronic activation of downstream signaling pathways. This persistent signaling activity contributes to the uncontrolled growth of uveal melanoma cells. As G protein-coupled receptors (GPCRs) play a crucial role in this process, screening libraries of GPCR inhibitors has been explored as a potential therapeutic avenue. These libraries comprise a vast array of compounds with the potential to block or modulate GPCR activity. Identifying specific inhibitors that can effectively interfere with the oncogenic signaling cascades triggered by the mutated Gαq protein is a critical step in developing targeted therapies for uveal melanoma. Methods: GPCR library high throughput screening employed isogenic cell lines expressing wild type or mutant (Q209L) GNAQ. This process utilized IP-One assays to identify compounds affecting Gq-coupled receptors by detecting inositol monophosphate accumulation. Subsequently, promising hits were evaluated for their impact on inhibiting the growth and proliferation of uveal melanoma cells. Further analysis involved Western blotting to assess their potential in promoting autophagy and apoptosis, inhibiting downstream signaling pathways, and a migration assay. Finally, ROS generation was assessed using flow cytometry. Results: Using isogenic cell lines expressing GNAQ gene wild type and the Q209L mutant, a high-throughput screening of GPCR libraries identified sertindole among 5 hits. Sertindole showed a dose-dependent inhibition of Q209L-mutated uveal melanoma cells, significantly reducing cell viability, proliferation, and migration. It also suppressed key signaling pathways, including the critical MAPK pathway, and activated autophagy and apoptosis, likely via ROS generation. These findings suggest sertindole's multifaceted approach in suppressing cancer in uveal melanoma. Conclusion: Our study highlights the potential therapeutic value of sertindole in GNAQ Q209L-mutated uveal melanoma. Further research is needed to evaluate the efficacy of sertindole in preclinical mouse models of uveal melanoma. Citation Format: Shelby Rheinschmidt, Taylor Bargenquast, Sydney Adamson, Trason Thode, Alexis Weston, Tithi Ghosh Halder, Serina Ng, Harini Tulasi, Brian Durbin, Justin Moser, Mohan Kaadige, Raffaella Soldi, Sunil Sharma. G-protein coupled receptor (GPCR) screening library identifies sertindole, an antipsychotic, as potential therapeutic option to target GNAQ mutation Q209L in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 671.

Arrestin-centred interactions at the membrane and their conformational determinants.

More than 30 years after their discovery, arrestins are recognised multiprotein scaffolds that play essential roles in G protein-coupled receptor (GPCR) regulation and signalling. Originally named for their capacity to hinder GPCR coupling to G proteins and facilitate receptor desensitisation, arrestins have emerged as key hubs for a myriad of other functions, including receptor internalisation and scaffolding of signalling complexes. Recent structural studies have started to provide snapshots of the complexes formed by GPCRs and arrestins, supporting a wealth of biochemical data delineating the molecular determinants of such interactions. Furthermore, biophysical techniques have also provided key information with regards to the basal and active conformations of arrestins, and how these are affected upon GPCR activation. Here, we review the most recent advances on our understanding of GPCR-arrestin complexes, from structure to interactions of arrestins with the lipid bilayer and other proteins. We also present an updated view on the development of tools to study the conformational flexibility of arrestins, with the potential to provide experimental data to describe the dynamic models of arrestin activation.

Glu1022.53-Mediated Early Conformational Changes in the Process of Light-Induced Green Cone Pigment Activation.

Ligand-triggered activation of G protein-coupled receptors (GPCRs) relies on the phenomenon of loose allosteric coupling, which involves conformational alterations spanning from the extracellular ligand-binding domain to the cytoplasmic region, where interactions with G proteins occur. During the GPCR activation process, several intermediate and equilibrium states orchestrate the movement of the flexible and rigid transmembrane (TM) segments of the GPCR. Monitoring early conformational changes is important in unraveling the structural intricacies of the loose allosteric coupling. Here, we focus on the lumi intermediate formed by thermal relaxation from the initial photointermediate, batho in primate green cone pigment (MG), a light-sensitive GPCR responsible for color vision. Our findings from light-induced Fourier transform infrared difference spectroscopy reveal its similarity with rhodopsin, which mediates twilight vision, specifically involving the flip motion of the β-ionone ring, the relaxation of the torsional structure of the retinal, and local perturbations in the α-helix upon lumi intermediate formation. Conversely, we observe a hydrogen bond modification specific to MG's protonated carboxylic acid, identifying its origin as Glu1022.53 situated in TM2. The weakening of the hydrogen bond strength at Glu1022.53 during the transition from the batho to the lumi intermediates corresponds to a slight outward movement of TM2. Additionally, within the X-ray crystal structure of the rhodopsin lumi intermediate, we note the relocation of the Met862.53 side chain in TM2, expanding the volume of the retinal binding pocket. Consequently, the position of 2.53 emerges as the early step in the conformational shift toward light-induced activation. Moreover, given the prevalence of IR-insensitive hydrophobic amino acids at position 2.53 in many rhodopsin-like GPCRs, including rhodopsin, the hydrogen bond alteration in the C═O stretching band at Glu1022.53 of MG can be used as a probe for tracing conformational changes during the GPCR activation process.

Specific pharmacological and Gi/o protein responses of some native GPCRs in neurons

G protein-coupled receptors (GPCRs) constitute the largest family of membrane proteins and are important drug targets. The discovery of drugs targeting these receptors and their G protein signaling properties are based on assays mainly performed with modified receptors expressed in heterologous cells. However, GPCR responses may differ in their native environment. Here, by using highly sensitive Gi/o sensors, we reveal specific properties of Gi/o protein-mediated responses triggered by GABAB, α2 adrenergic and cannabinoid CB1 receptors in primary neurons, different from those in heterologous cells. These include different profiles in the Gi/o protein subtypes-mediated responses, and differences in the potencies of some ligands even at similar receptor expression levels. Altogether, our results show the importance of using biosensors compatible with primary cells for evaluating the activities of endogenous GPCRs in their native environment.

Direct interrogation of context-dependent GPCR activity with a universal biosensor platform

G protein-coupled receptors (GPCRs) are the largest family of druggable proteins encoded in the human genome, but progress in understanding and targeting them is hindered by the lack of tools to reliably measure their nuanced behavior in physiologically relevant contexts. Here, we developed a collection of compact ONE vector G-protein Optical (ONE-GO) biosensor constructs as a scalable platform that can be conveniently deployed to measure G-protein activation by virtually any GPCR with high fidelity even when expressed endogenously in primary cells. By characterizing dozens of GPCRs across many cell types like primary cardiovascular cells or neurons, we revealed insights into the molecular basis for G-protein coupling selectivity of GPCRs, pharmacogenomic profiles of anti-psychotics on naturally occurring GPCR variants, and G-protein subtype signaling bias by endogenous GPCRs depending on cell type or upon inducing disease-like states. In summary, this open-source platform makes the direct interrogation of context-dependent GPCR activity broadly accessible.

Probing Activation and Conformational Dynamics of the Vesicle-Reconstituted β2 Adrenergic Receptor at the Single-Molecule Level.

G-protein-coupled receptors (GPCRs) are structurally flexible membrane proteins that mediate a host of physiological responses to extracellular ligands like hormones and neurotransmitters. Fine features of their dynamic structural behavior are hypothesized to encode the functional plasticity seen in GPCR activity, where ligands with different efficacies can direct the same receptor toward different signaling phenotypes. Although the number of GPCR crystal structures is increasing, the receptors are characterized by complex and poorly understood conformational landscapes. Therefore, we employed a fluorescence microscopy assay to monitor conformational dynamics of single β2 adrenergic receptors (β2ARs). To increase the biological relevance of our findings, we decided not to reconstitute the receptor in detergent micelles but rather lipid membranes as proteoliposomes. The conformational dynamics were monitored by changes in the intensity of an environmentally sensitive boron-dipyrromethene (BODIPY 493/503) fluorophore conjugated to an endogenous cysteine (located at the cytoplasmic end of the sixth transmembrane helix of the receptor). Using total internal reflection fluorescence microscopy (TIRFM) and a single small unilamellar liposome assay that we previously developed, we followed the real-time dynamic properties of hundreds of single β2ARs reconstituted in a native-like environment─lipid membranes. Our results showed that β2AR-BODIPY fluctuates between several states of different intensity on a time scale of seconds, compared to BODIPY-lipid conjugates that show almost entirely stable fluorescence emission in the absence and presence of the full agonist BI-167107. Agonist stimulation changes the β2AR dynamics, increasing the population of states with higher intensities and prolonging their durations, consistent with bulk experiments. The transition density plot demonstrates that β2AR-BODIPY, in the absence of the full agonist, interconverts between states of low and moderate intensity, while the full agonist renders transitions between moderate and high-intensity states more probable. This redistribution is consistent with a mechanism of conformational selection and is a promising first step toward characterizing the conformational dynamics of GPCRs embedded in a lipid bilayer.

GPCRs involved in metabolic diseases: pharmacotherapeutic development updates.

G protein-coupled receptors (GPCRs) are expressed in a variety of cell types and tissues, and activation of GPCRs is involved in enormous metabolic pathways, including nutrient synthesis, transportation, storage or insulin sensitivity, etc. This review intends to summarize the regulation of metabolic homeostasis and mechanisms by a series of GPCRs, such as GPR91, GPR55, GPR119, GPR109a, GPR142, GPR40, GPR41, GPR43 and GPR120. With deep understanding of GPCR's structure and signaling pathways, it is attempting to uncover the role of GPCRs in major metabolic diseases, including metabolic syndrome, diabetes, dyslipidemia and nonalcoholic steatohepatitis, for which the global prevalence has risen during last two decades. An extensive list of agonists and antagonists with their chemical structures in a nature of small molecular compounds for above-mentioned GPCRs is provided as pharmacologic candidates, and their preliminary data of preclinical studies are discussed. Moreover, their beneficial effects in correcting abnormalities of metabolic syndrome, diabetes and dyslipidemia are summarized when clinical trials have been undertaken. Thus, accumulating data suggest that these agonists or antagonists might become as new pharmacotherapeutic candidates for the treatment of metabolic diseases.

Scaling up Functional Analyses of the G Protein-Coupled Receptor Rhodopsin.

Eukaryotic cells use G protein-coupled receptors (GPCRs) to convert external stimuli into internal signals to elicit cellular responses. However, how mutations in GPCR-coding genes affect GPCR activation and downstream signaling pathways remain poorly understood. Approaches such as deep mutational scanning show promise in investigations of GPCRs, but a high-throughput method to measure rhodopsin activation has yet to be achieved. Here, we scale up a fluorescent reporter assay in budding yeast that we engineered to study rhodopsin's light-activated signal transduction. Using this approach, we measured the mutational effects of over 1200 individual human rhodopsin mutants, generated by low-frequency random mutagenesis of the GPCR rhodopsin (RHO) gene. Analysis of the data in the context of rhodopsin's three-dimensional structure reveals that transmembrane helices are generally less tolerant to mutations compared to flanking helices that face the lipid bilayer, which suggest that mutational tolerance is contingent on both the local environment surrounding specific residues and the specific position of these residues in the protein structure. Comparison of functional scores from our screen to clinically identified rhodopsin disease variants found many pathogenic mutants to be loss of function. Lastly, functional scores from our assay were consistent with a complex counterion mechanism involved in ligand-binding and rhodopsin activation. Our results demonstrate that deep mutational scanning is possible for rhodopsin activation and can be an effective method for revealing properties of mutational tolerance that may be generalizable to other transmembrane proteins.

Engineering, applications, and future perspectives of GPCR-based genetically encoded fluorescent indicators for neuromodulators.

This review explores the evolving landscape of G-protein-coupled receptor (GPCR)-based genetically encoded fluorescent indicators (GEFIs), with a focus on their development, structural components, engineering strategies, and applications. We highlight the unique features of this indicator class, emphasizing the importance of both the sensing domain (GPCR structure and activation mechanism) and the reporting domain (circularly permuted fluorescent protein (cpFP) structure and fluorescence modulation). Further, we discuss indicator engineering approaches, including the selection of suitable cpFPs and expression systems. Additionally, we showcase the diversity and flexibility of their application by presenting a summary of studies where such indicators were used. Along with all the advantages, we also focus on the current limitations as well as common misconceptions that arise when using these indicators. Finally, we discuss future directions in indicator engineering, including strategies for screening with increased throughput, optimization of the ligand-binding properties, structural insights, and spectral diversity.

GRK2 mediated degradation of SAV1 initiates hyperplasia of fibroblast-like synoviocytes in rheumatoid arthritis

Hyperplasia and migration of fibroblast-like synoviocytes (FLSs) are the key drivers in the pathogenesis of rheumatoid arthritis (RA) and joint destruction. Abundant Yes-associated protein (YAP), which is a powerful transcription co-activator for proliferative genes, was observed in the nucleus of inflammatory FLSs with unknown upstream mechanisms. Using Gene Expression Omnibus database analysis, it was found that Salvador homolog-1 (SAV1), the pivotal negative regulator of the Hippo-YAP pathway, was slightly downregulated in RA synovium. However, SAV1 protein expression is extremely reduced. Subsequently, it was revealed that SAV1 is phosphorylated, ubiquitinated, and degraded by interacting with an important serine-threonine kinase, G protein-coupled receptor (GPCR) kinase 2 (GRK2), which was predominately upregulated by GPCR activation induced by ligands such as prostaglandin E2 (PGE2) in RA. This process further contributes to the decreased phosphorylation, nuclear translocation, and transcriptional potency of YAP, and leads to aberrant FLSs proliferation. Genetic depletion of GRK2 or inhibition of GRK2 by paroxetine rescued SAV1 expression and restored YAP phosphorylation and finally inhibited RA FLSs proliferation and migration. Similarly, paroxetine treatment effectively reduced the abnormal proliferation of FLSs in a rat model of collagen-induced arthritis which was accompanied by a significant improvement in clinical manifestations. Collectively, these results elucidate the significance of GRK2 regulation of Hippo-YAP signaling in FLSs proliferation and migration and the potential application of GRK2 inhibition in the treatment of FLSs-driven joint destruction in RA.

Characterization of a novel RNAi yeast insecticide that silences mosquito 5-HT1 receptor genes

G protein-coupled receptors (GPCRs), which regulate numerous intracellular signaling cascades that mediate many essential physiological processes, are attractive yet underexploited insecticide targets. RNA interference (RNAi) technology could facilitate the custom design of environmentally safe pesticides that target GPCRs in select target pests yet are not toxic to non-target species. This study investigates the hypothesis that an RNAi yeast insecticide designed to silence mosquito serotonin receptor 1 (5-HTR1) genes can kill mosquitoes without harming non-target arthropods. 5-HTR.426, a Saccharomyces cerevisiae strain that expresses an shRNA targeting a site specifically conserved in mosquito 5-HTR1 genes, was generated. The yeast can be heat-inactivated and delivered to mosquito larvae as ready-to-use tablets or to adult mosquitoes using attractive targeted sugar baits (ATSBs). The results of laboratory and outdoor semi-field trials demonstrated that consumption of 5-HTR.426 yeast results in highly significant mortality rates in Aedes, Anopheles, and Culex mosquito larvae and adults. Yeast consumption resulted in significant 5-HTR1 silencing and severe neural defects in the mosquito brain but was not found to be toxic to non-target arthropods. These results indicate that RNAi insecticide technology can facilitate selective targeting of GPCRs in intended pests without impacting GPCR activity in non-targeted organisms. In future studies, scaled production of yeast expressing the 5-HTR.426 RNAi insecticide could facilitate field trials to further evaluate this promising new mosquito control intervention.

Phosphorylation Sites of the Gastric Inhibitory Polypeptide Receptor (GIPR) Revealed by Trapped-Ion-Mobility Spectrometry Coupled to Time-of-Flight Mass Spectrometry (TIMS-TOF MS).

The gastric inhibitory polypeptide receptor (GIPR), a G protein-coupled receptor (GPCR) that regulates glucose metabolism and insulin secretion, is a target for the development of therapeutic agents to address type 2 diabetes and obesity. Signal transduction processes mediated by GPCR activation typically result in receptor phosphorylation, but very little is known about GIPR phosphorylation. Mass spectrometry (MS) is a powerful tool for detecting phosphorylation and other post-translational modifications of proteins and for identifying modification sites. However, applying MS methods to GPCRs is challenging because the native expression levels are low and the hydrophobicity of these proteins complicates isolation and enrichment. Here we use a widely available technique, trapped-ion-mobility spectrometry coupled to time-of-flight mass spectrometry (TIMS-TOF MS), to characterize the phosphorylation status of the GIPR. We identified eight serine residues that are phosphorylated, one in an intracellular loop and the remainder in the C-terminal domain. Stimulation with the native agonist GIP enhanced phosphorylation at four of these sites. For comparison, we evaluated tirzepatide (TZP), a dual agonist of the glucagon-like peptide-1 (GLP-1) receptor and the GIPR that has recently been approved for the treatment of type 2 diabetes. Stimulation with TZP enhanced phosphorylation at the same four sites that were enhanced with GIP; however, TZP also enhanced phosphorylation at a fifth site that is unique to this synthetic agonist. This work establishes an important and accessible tool for the characterization of signal transduction via the GIPR and reveals an unanticipated functional difference between GIP and TZP.

A rapid, tag-free way to purify functional GPCRs

G protein-coupled receptors (GPCRs) play diverse signaling roles and represent major pharmaceutical targets. Consequently, they are the focus of intense study, and numerous advances have been made in their handling and analysis. However, a universal way to purify GPCRs has remained elusive, in part because of their inherent instability when isolated from cells. To address this, we have developed a general, rapid, and tag-free way to purify GPCRs. The method uses short peptide analogs of the Gα subunit C terminus (Gα-CT) that are attached to chromatography beads (Gα-CT resin). Because the Gα-CT peptides bind active GPCRs with high affinity, the Gα-CT resin selectively purifies only active functional receptors. We use this method to purify both rhodopsin and the β2-adrenergic receptor and show they can be purified in either active conformations or inactive conformations, simply by varying elution conditions. While simple in concept-leveraging the conserved GPCR-Gα-CT binding interaction for the purpose of GPCR purification-we think this approach holds excellent potential to isolate functional receptors for a myriad of uses, from structural biology to proteomics.

β-arrestin1 is an E3 ubiquitin ligase adaptor for substrate linear polyubiquitination

G protein-coupled receptor (GPCR) signaling and trafficking are regulated by multiple mechanisms, including posttranslational modifications such as ubiquitination by E3 ubiquitin ligases. E3 ligases have been linked to agonist-stimulated ubiquitination of GPCRs via simultaneous binding to βarrestins. In addition, βarrestins have been suggested to assist E3 ligases for ubiquitination of key effector molecules, yet mechanistic insight is lacking. Here, we developed an invitro reconstituted system and show that βarrestin1 (βarr1) serves as an adaptor between the effector protein signal-transducing adaptor molecule 1 (STAM1) and the E3 ligase atrophin-interacting protein 4. Via mass spectrometry, we identified seven lysine residues within STAM1 that are ubiquitinated and several types of ubiquitin linkages. We provide evidence that βarr1 facilitates the formation of linear polyubiquitin chains at lysine residue 136 on STAM1. This lysine residue is important for stabilizing the βarr1:STAM1 interaction in cells following GPCR activation. Our study identifies atrophin-interacting protein 4 as only the second E3 ligase known to conjugate linear polyubiquitin chains and a possible role for linear ubiquitin chains in GPCR signaling and trafficking.

Identification of a carbohydrate recognition motif of purinergic receptors.

As a major class of biomolecules, carbohydrates play indispensable roles in various biological processes. However, it remains largely unknown how carbohydrates directly modulate important drug targets, such as G-protein coupled receptors (GPCRs). Here, we employed P2Y purinoceptor 14 (P2Y14), a drug target for inflammation and immune responses, to uncover the sugar nucleotide activation of GPCRs. Integrating molecular dynamics simulation with functional study, we identified the uridine diphosphate (UDP)-sugar-binding site on P2Y14, and revealed that a UDP-glucose might activate the receptor by bridging the transmembrane (TM) helices 2 and 7. Between TM2 and TM7 of P2Y14, a conserved salt bridging chain (K2.60-D2.64-K7.35-E7.36 [KDKE]) was identified to distinguish different UDP-sugars, including UDP-glucose, UDP-galactose, UDP-glucuronic acid, and UDP-N-acetylglucosamine. We identified the KDKE chain as a conserved functional motif of sugar binding for both P2Y14 and P2Y purinoceptor 12 (P2Y12), and then designed three sugar nucleotides as agonists of P2Y12. These results not only expand our understanding for activation of purinergic receptors but also provide insights for the carbohydrate drug development for GPCRs.

Ageing influences detrusor contractions to prostaglandin, angiotensin, histamine and 5-HT (serotonin), independent to the Rho kinase and extracellular calcium pathways

Ageing is associated with deteriorating urinary bladder function and an increasing prevalence of disorders such as underactive bladder. There are suggestions that G protein-coupled receptor (GPCR) second messenger pathways are altered during ageing, rather than the receptor proteins themselves. The aim of this study was to identify age-related variations in GPCR activation systems in urinary bladder smooth muscle (detrusor). Isolated porcine detrusor strips were mounted in organ baths and contractile responses induced by receptor agonists were assessed and compared between juvenile (6 months) and adult (2 years) animals. The effects of drugs disrupting intracellular calcium signalling were also studied. Adult tissue was far more sensitive to stimulation by 5-hydroxytryptamine (42% greater increase than juvenile), prostaglandin-E2 (26% greater increase), and angiotensin-II (39% greater increase), however less sensitive to histamine. Although nifedipine and Y-27632 impacted the contraction to all agonists, there were no significant differences between juvenile and adult detrusor. Impairment of IP3-mediated calcium release by 2-aminoethyl diphenylborinate had no effect on any contractile activity, except for neurokinin-A which inhibited both juvenile and adult detrusor, and prostaglandin-E2 which inhibited juvenile. Carbachol, histamine, 5-hydroxytryptamine, and angiotensin-II were not affected by the application of 2-aminoethyl diphenylborinate. In conclusion, the contractile responses to all the GPCR agonists involved extracellular calcium influx and calcium sensitisation, but for prostaglandin-E2 the dependence on calcium from intracellular sources was greater in the younger animals.

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

β-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. β-arrestins link agonist-stimulated GPCRs to downstream signaling partners, such as the c-Raf-MEK1-ERK1/2 cascade leading to ERK1/2 activation. β-arrestins have been thought to transduce signals solely via passive scaffolding by facilitating the assembly of multiprotein signaling complexes. Recently, however, β-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 β-arrestins. Here, we demonstrate that β-arrestins play an active role in allosterically modulating ERK kinase activity in vitro and within intact cells. Specifically, we show that β-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 β-arrestins do so. Furthermore, we find that allosteric stimulation of dually phosphorylated ERK2 by active-state β-arrestin 2 is more robust than by active-state β-arrestin 1, highlighting differential capacities of β-arrestin isoforms to regulate effector signaling pathways downstream of GPCRs. In summary, our study provides strong evidence for a new paradigm in which β-arrestins function as active "catalytic" scaffolds to allosterically unlock the enzymatic activity of signaling components downstream of GPCR activation.