G Protein-coupled Receptors Targets Research Articles

Fragment-Based Computational Method for Designing GPCR Ligands.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors, which is arguably the most important family of drug target. With the technology breakthroughs in X-ray crystallography and cryo-electron microscopy, more than 300 GPCR-ligand complex structures have been publicly reported since 2007, covering about 60 unique GPCRs. Such abundant structural information certainly will facilitate the structure-based drug design by targeting GPCRs. In this study, we have developed a fragment-based computational method for designing novel GPCR ligands. We first extracted the characteristic interaction patterns (CIPs) on the binding interfaces between GPCRs and their ligands. The CIPs were used as queries to search the chemical fragments derived from GPCR ligands, which were required to form similar interaction patterns with GPCR. Then, the selected chemical fragments were assembled into complete molecules by using the AutoT&T2 software. In this work, we chose β-adrenergic receptor (β-AR) and muscarinic acetylcholine receptor (mAChR) as the targets to validate this method. Based on the designs suggested by our method, samples of 63 compounds were purchased and tested in a cell-based functional assay. A total of 15 and 22 compounds were identified as active antagonists for β2-AR and mAChR M1, respectively. Molecular dynamics simulations and binding free energy analysis were performed to explore the key interactions (e.g., hydrogen bonds and π-π interactions) between those active compounds and their target GPCRs. In summary, our work presents a useful approach to the de novo design of GPCR ligands based on the relevant 3D structural information.

Toward G protein-coupled receptor structure-based drug design using X-ray lasers.

Rational structure-based drug design (SBDD) relies on the availability of a large number of co-crystal structures to map the ligand-binding pocket of the target protein and use this information for lead-compound optimization via an iterative process. While SBDD has proven successful for many drug-discovery projects, its application to G protein-coupled receptors (GPCRs) has been limited owing to extreme difficulties with their crystallization. Here, a method is presented for the rapid determination of multiple co-crystal structures for a target GPCR in complex with various ligands, taking advantage of the serial femtosecond crystallography approach, which obviates the need for large crystals and requires only submilligram quantities of purified protein. The method was applied to the human β2-adrenergic receptor, resulting in eight room-temperature co-crystal structures with six different ligands, including previously unreported structures with carvedilol and propranolol. The generality of the proposed method was tested with three other receptors. This approach has the potential to enable SBDD for GPCRs and other difficult-to-crystallize membrane proteins.

Short-chain fatty acids and microbiota metabolites attenuate ghrelin receptor signaling.

The gastrointestinal microbiota is emerging as a unique and inexhaustible source for metabolites with potential to modulate G-protein coupled receptors (GPCRs). The ghrelin receptor [growth hormone secretagogue receptor (GHSR)-1a] is a GPCR expressed throughout both the gut and the brain and plays a crucial role in maintaining energy balance, metabolism, and the central modulation of food intake, motivation, reward, and mood. To date, few studies have investigated the potential of the gastrointestinal microbiota and its metabolites to modulate GPCR signaling. Here we investigate the ability of short-chain fatty acids (SCFAs), lactate, and different bacterial strains, including Bifidobacterium and Lactobacillus genera, to modulate GHSR-1a signaling. We identify, for what is to our knowledge the first time, a potent effect of microbiota-derived metabolites on GHSR-1a signaling with potential significant consequences for host metabolism and physiology. We show that SCFAs, lactate, and bacterial supernatants are able to attenuate ghrelin-mediated signaling through the GHSR-1a. We suggest a novel route of communication between the gut microbiota and the host via modulation of GHSR-1a receptor signaling. Together, this highlights the emerging therapeutic potential in the exploration of the microbiota metabolome in the specific targeting of key GPCRs, with pleiotropic actions that span both the CNS and periphery.-Torres-Fuentes, C., Golubeva, A. V., Zhdanov, A. V., Wallace, S., Arboleya, S., Papkovsky, D. B., El Aidy, S., Ross, P., Roy, B. L., Stanton, C., Dinan, T. G., Cryan, J. F., Schellekens, H. Short-chain fatty acids and microbiota metabolites attenuate ghrelin receptor signaling.

Effects of Post-translational Modifications on Membrane Localization and Signaling of Prostanoid GPCR-G Protein Complexes and the Role of Hypoxia.

G protein-coupled receptors (GPCRs) play a pivotal role in the adaptive responses to cellular stresses such as hypoxia. In addition to influencing cellular gene expression profiles, hypoxic microenvironments can perturb membrane protein localization, altering GPCR effector scaffolding and altering downstream signaling. Studies using proteomics approaches have revealed significant regulation of GPCR and G proteins by their state of post-translational modification. The aim of this review is to examine the effects of post-translational modifications on membrane localization and signaling of GPCR-G protein complexes, with an emphasis on vascular prostanoid receptors, and to highlight what is known about the effect of cellular hypoxia on these mechanisms. Understanding post-translational modifications of protein targets will help to define GPCR targets in treatment of disease, and to inform research into mechanisms of hypoxic cellular responses.

The role of water and protein flexibility in the structure-based virtual screening of allosteric GPCR modulators: an mGlu5 receptor case study

Stabilizing unique receptor conformations, allosteric modulators of G-protein coupled receptors (GPCRs) might open novel treatment options due to their new pharmacological action, their enhanced specificity and selectivity in both binding and signaling. Ligand binding occurs at intrahelical allosteric sites and involves significant induced fit effects that include conformational changes in the local protein environment and water networks. Based on the analysis of available crystal structures of metabotropic glutamate receptor 5 (mGlu5) we investigated these effects in the binding of mGlu5 receptor negative allosteric modulators. A large set of retrospective virtual screens revealed that the use of multiple protein structures and the inclusion of selected water molecules improves virtual screening performance compared to conventional docking strategies. The role of water molecules and protein flexibility in ligand binding can be taken into account efficiently by the proposed docking protocol that provided reasonable enrichment of true positives. This protocol is expected to be useful also for identifying intrahelical allosteric modulators for other GPCR targets.

Specific miRNA-G Protein-Coupled Receptor Networks Regulate Sox9a/Sox9b Activities to Promote Gonadal Rejuvenation in Zebrafish.

Fertility and endocrine function rely on a tightly regulated synchronicity within the hypothalamic-pituitary-gonadal axis, for which the sex gonad serves as the primary source of sex steroid hormones and germ cells. To maintain hormonal stasis and fertility throughout the lifespan, inducing gonadal stem cell renewal is an attractive strategy. The follicle-stimulating hormone/cAMP/MAPK/Sox9 signaling axis and its regulated specific miRNAs are thought to regulate vertebrate gonadal development and sex differentiation, yet the regulatory networks are largely unknown. By genome-wide transcriptome mining and gonadal microinjections, we identify two G protein-coupled receptor (GPCR)-regulatory circuits: miR430a-Sox9a in the testis and miR218a-Sox9b in the ovary. Coinjection of a Sox9a-miR430a mixture promotes spermatogenesis, whereas Sox9b-miR218a mixture increases primordial ovarian follicles. Coimmunoprecipitation and mass spectrometry indicate that the two mixtures differentially modulate Sox9a/Sox9b multiple covalent modifications. We further reveal that miR430a and Sox9a synergistically activate testicular protein kinase C (PKC)/Akt signaling, whereas the miR218a and Sox9b mixture constrains ovary PKC/Akt signaling. pMIR-GFP reporter assay demonstrate that miR430a and miR218a target the 3' untranslated region (UTR) of four GPCR targets (lgr4, grk5l, grk4, and grp157). Knockdown of these GPCR genes or two Sox9 genes alters miR430a and miR218a regulation in the above gonad-specific PKC and Akt signaling pathways. These results establish two specific miRNA-GPCR-Sox9 networks and provide mechanistic insight into gonadal differentiation and rejuvenation. Stem Cells 2019;37:1189-1199.

A cell–cell interaction format for selection of high-affinity antibodies to membrane proteins

Generating and improving antibodies and peptides that bind specifically to membrane protein targets such as ion channels and G protein-coupled receptors (GPCRs) can be challenging using established selection methods. Current strategies are often limited by difficulties in the presentation of the antigen or the efficiency of the selection process. Here, we report a method for obtaining antibodies specific for whole cell membrane-associated antigens which combines a cell-cell interaction format based on yeast display technology with fluorescence-activated cell sorting of dual fluorescent complexes. Using this method, we were able to direct the affinity maturation of an antagonist antibody specific for the proton-gated ion channel ASIC1a and showed that both the affinity and potency were improved. We were also able to use this method to do kinetic selections to generate clones with better dissociation profiles. In addition, this method was employed successfully to handle the difficult problem of selecting antibodies specific to a GPCR target, the mu-opioid receptor.

Abstract 2053: Targeting GRK2 increases chemotherapy-induced apoptosis in medulloblastoma

Abstract Background: G protein receptor kinase-2 (GRK2; ADRBK1) belongs to the family of G protein-coupled receptor (GPCR) kinases (GRKs). GRK2 phosphorylates GPCR and non-GPCR targets including cell surface receptors, cytoskeletal and mitochondrial proteins and transcription factors. It also functions in embryonic development, heart function, metabolism, and some cancer models. Here we hypothesized that targeting GRK2 will sensitizes cancer cells to therapy. We tested this in medulloblastoma, the most common malignant brain tumor in children. Methods: siRNA transfections, shRNA viral infections, SDS-PAGE, western blotting, flow cytometry. Cells: UW228 and DAOY medulloblastoma. Results: We found that GRK2 inhibition promotes anti-tumor effects of chemotherapeutic drugs cisplatin and etoposide in UW228 and DAOY medulloblastoma cell lines. GRK2 silencing increased apoptosis induced by cisplatin and etoposide and GRK2 overexpression decreased apoptosis induced by these drugs in UW228 and DAOY. Time dependent studies revealed that GRK2 overexpression delayed cisplatin- and etoposide-induced apoptosis measured by the induction of Annexin V positive population and by PARP-1 cleavage. GRK2 mutants, GRK2 K220R (kinase deficient mutant) and GRK2 S670A (cannot be phosphorylated) were similar to wildtype GRK2 in delaying the cisplatin and etoposide induced apoptosis. GRK2 knockdown antagonized serum induced AKT and ERK phosphorylation in both lines whereas GRK2 overexpression promoted phosphorylation, suggesting that GRK2 is important in medulloblastoma survival pathways. Conclusions: These results demonstrate that targeting GRK2 enhances cisplatin- and etoposide-induced apoptosis in medulloblastoma cell lines and suggests that GRK2 inhibitors may enhance responsiveness of medulloblastoma to chemotherapy. Citation Format: Anup S. Pathania, Xiuhai Ren, Min Mahdi, Gregory M. Shackleford, Anat Erdreich-Epstein. Targeting GRK2 increases chemotherapy-induced apoptosis in medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2053.

Probing Multi-Target Action of Phlorotannins as New Monoamine Oxidase Inhibitors and Dopaminergic Receptor Modulators with the Potential for Treatment of Neuronal Disorders.

Modulation of multiple protein targets with a single compound is essential for the effective treatment of central nervous system disorders. In our previous G protein-coupled receptor (GPCR) cell-based study, a selective human monoamine oxidase (hMAO)-A inhibitor, eckol, stimulated activity of dopamine D3 and D4 receptors. This result led to our interest in marine phlorotannin-mediated modulation of hMAO enzymes and related GPCRs in neuronal disorders. Here, we evaluate the multi-target effects of phloroglucinol, phlorofucofuroeckol-A (PFF-A), and dieckol by screening their modulatory activity against hMAO-A and -B and various neuronal GPCRs. Among the tested phlorotannins, PFF-A showed the strongest inhibitory activity against both hMAO isoforms, with higher selectivity toward hMAO-B than hMAO-A. Enzyme kinetics and docking data revealed that PFF-A noncompetitively acts on hMAOs into the alternative binding pocket of enzymes with allosteric functions. In a functional assay for GPCR screening, dieckol and PFF-A exhibited a multi-target combination of D3R/D4R agonism and D1/5HT1A/NK1 antagonism. In particular, they effectively stimulated D3R and D4R, compared to other GPCRs. Docking analysis confirmed that dieckol and PFF-A successfully docked into the conserved active sites of D3R and D4R and interacted with aspartyl and serine residues in the orthosteric binding pockets of the respective receptors. Based on our experimental and computational data, we established the structure-activity relationship between tested phlorotannins and target proteins, including hMAOs and GPCRs. Our current findings suggest that hMAO inhibitors dieckol and PFF-A, major phlorotannins of edible brown algae with multi-action on GPCRs, are potential agents for treatment of psychological disorders and Parkinson’s disease.

Don't trust the datasheet: a validation of commercial G alpha i/o primary antibodies for Western blotting

Primary antibodies are an integral part of Western blotting but validation of the specificity and sensitivity of antibodies by suppliers is extremely lacking. Multiple examples of this are evident in the G alpha i/o family of proteins. G alpha proteins are important signal transducing molecules for both endogenous cellular function and drug action that are divided into four families: Gs, Gi/o, Gq/11, and G12/13. These four families of proteins interact with the nearly 1000 G protein coupled receptors (GPCRs) in the human body, the largest and most diverse group of receptors. Because of the diversity of these receptors, around one third of all drugs on the market target GPCRs, which, in part, utilize different G alpha subunit proteins to evoke their downstream signaling and physiological responses. Therefore, it is important that researchers have tools; e.g., selective antibodies, that can clearly define how G alpha proteins produce their effects associated with a receptor and its ligand. Multiple commercially available G alpha protein antibodies have been employed for relative G protein quantification, but they have not been adequately verified for both their specificity and sensitivity. In this study, BL‐21 DE3 RIL competent cells were used to express and purify five protein members of the Gi/o family of G alpha proteins. Gi1, Gi2, Gi3, Go, and Gz proteins were purified using an AKTA FPLC system and a histidine trap purification column. Western blots using these proteins and multiple Gi/o primary antibodies from different vendors were used to determine the specificity of the antibodies for their advertised protein. Our results showed antibodies that were successful in binding specifically to their target and antibodies that either did not bind to the protein of interest or expressed binding to multiple members of the Gi/o family despite claims that it was selective in targeting a single protein. After determination of specificity, purified proteins were used in a 1:2 dilution series to determine the linear range of detection for each antibody that was deemed specific for a single protein. This linear range was then used for determination of sensitivity and absolute quantitation of Gi/o proteins in Sprague‐Dawley rat brain tissue, specifically punches from the paraventricular nucleus and cortex. Together, the results of these studies indicate that certain antibodies, which are available from commercial vendors for use in Western blot assays, may not be selective for a specific Gi/o subunit protein as advertised. Thus, it is essential that investigators be sure to verify that their antibody is selective and specific for their target protein in their own hands, to ensure that the use of the antibody in subsequent studies is valid.Support or Funding InformationNIH 5P30GM106392 to DK and R01GM097350 to SKThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Specific TBC Domain‐Containing Proteins Control the ER‐Golgi‐Plasma Membrane Trafficking of GPCRs

G protein‐coupled receptors (GPCRs) constitute the largest superfamily of cell‐surface signaling proteins. However, the molecular mechanisms underlying their cell surface delivery after synthesis remain poorly understood. Here we screen the TBC domain‐containing proteins, putative Rab GTPase‐activating proteins (GAPs), in the intracellular trafficking of GPCRs and identify several TBC proteins that activity‐dependently regulate the anterograde transport, en route from the endoplasmic reticulum to the Golgi or from the Golgi to the cell surface, of several prototypic GPCR members without affecting other plasma membrane proteins. We also show that TBC1D6 functions as a GAP for Rab26, physically associates with Rab26, and attenuates Rab26 interaction with GPCRs. Furthermore, both overexpression and depletion of TBC1D6 inhibit the post‐Golgi traffic of GPCRs. These data demonstrate for the first time the important roles of the TBC proteins in forward trafficking of nascent GPCRs and reveal novel regulatory mechanisms of GPCR targeting to the functional destination.Support or Funding InformationNIH R01GM118915This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Development of Enhanced Conformational Sampling Methods for GPCRs

G protein‐coupled receptors (GPCRs) are known to possess multiple active conformational states in nature, however, studying these conformations is extremely challenging as majority of the functionally important conformations have high energy compared to the lowest energy conformation. We are developing a method called Enhanced Conformational Markov‐state Sampling in Membrane BiLayer Environment (EnCoMSeMBLE) that would enhance our search of the conformational landscape of GPCRs and that can be applied to a‐helical transmembrane proteins in general. It enables a level of conformational sampling not achievable by classical or accelerated molecular dynamics (MD) simulations or Markov‐State Models (MSM). This method combines brute force conformational sampling of helix‐helix interactions in the membrane with MD simulations and Markov‐State modeling to identify functionally important conformations. This method is being tested by application to the Glucagon Like peptide‐1 receptor (GLP1‐R), a class B GPCR, and the muscarinic acetylcholine M2 receptor, a class A GPCR, both of which have been crystallized in inactive as well as active conformations. The comparison of the activation landscapes of class A and class B GPCRs is beginning to provide key similarities and differences in activation across these very distinct GPCRs. This detailed understanding of the GPCR activation, enabled by our method, complements major structural biology efforts underway targeting GPCRs, where it is very challenging to map out the structural landscape and activation pathways of GPCRs.Support or Funding InformationBUILD PODER is supported through a grant from the National Institutes of General Medical Sciences (NIGMS), grants # # 8TL4GM11897This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

G protein-coupled receptor trafficking and signaling: new insights into the enteric nervous system.

G protein-coupled receptors (GPCRs) are essential for the neurogenic control of gastrointestinal (GI) function and are important and emerging therapeutic targets in the gut. Detailed knowledge of both the distribution and functional expression of GPCRs in the enteric nervous system (ENS) is critical toward advancing our understanding of how these receptors contribute to GI function during physiological and pathophysiological states. Equally important, but less well defined, is the complex relationship between receptor expression, ligand binding, signaling, and trafficking within enteric neurons. Neuronal GPCRs are internalized following exposure to agonists and under pathological conditions, such as intestinal inflammation. However, the relationship between the intracellular distribution of GPCRs and their signaling outputs in this setting remains a "black box". This review will briefly summarize current knowledge of agonist-evoked GPCR trafficking and location-specific signaling in the ENS and identifies key areas where future research could be focused. Greater understanding of the cellular and molecular mechanisms involved in regulating GPCR signaling in the ENS will provide new insights into GI function and may open novel avenues for therapeutic targeting of GPCRs for the treatment of digestive disorders.

Structural biology of G protein-coupled receptor signaling complexes.

G protein-coupled receptors (GPCRs) constitute the largest family of cell surface receptors that mediate numerous cell signaling pathways, and are targets of more than one-third of clinical drugs. Thanks to the advancement of novel structural biology technologies, high-resolution structures of GPCRs in complex with their signaling transducers, including G-protein and arrestin, have been determined. These 3D complex structures have significantly improved our understanding of the molecular mechanism of GPCR signaling and provided a structural basis for signaling-biased drug discovery targeting GPCRs. Here we summarize structural studies of GPCR signaling complexes with G protein and arrestin using rhodopsin as a model system, and highlight the key features of GPCR conformational states in biased signaling including the sequence motifs of receptor TM6 that determine selective coupling of G proteins, and the phosphorylation codes of GPCRs for arrestin recruitment. We envision the future of GPCR structural biology not only to solve more high-resolution complex structures but also to show stepwise GPCR signaling complex assembly and disassembly and dynamic process of GPCR signal transduction.

Structural organization of a major neuronal G protein regulator, the RGS7-Gβ5-R7BP complex.

Signaling by the G-protein-coupled receptors (GPCRs) plays fundamental role in a vast number of essential physiological functions. Precise control of GPCR signaling requires action of regulators of G protein signaling (RGS) proteins that deactivate heterotrimeric G proteins. RGS proteins are elaborately regulated and comprise multiple domains and subunits, yet structural organization of these assemblies is poorly understood. Here, we report a crystal structure and dynamics analyses of the multisubunit complex of RGS7, a major regulator of neuronal signaling with key roles in controlling a number of drug target GPCRs and links to neuropsychiatric disease, metabolism, and cancer. The crystal structure in combination with molecular dynamics and mass spectrometry analyses reveals unique organizational features of the complex and long-range conformational changes imposed by its constituent subunits during allosteric modulation. Notably, several intermolecular interfaces in the complex work in synergy to provide coordinated modulation of this key GPCR regulator.

Network pharmacology study of Xiaoxuming Decoction based on vasodilatory and vasoconstrictory related GPCR targets

In this study, bioinformatics methods such as molecular docking and network pharmacology were adopted to establish Xiaoxuming Decoction (XXMD) "compound-vasodilatory and vasoconstrictory related G protein-coupled receptors (GPCR) targets" network, then the vascular function regulatory effective components and the potential targets of XXMD were analyzed. Based on the XXMD herb sources, the chemical structures of the compounds were retrieved from the national scientific data sharing platform for population and health pharmaceutical information center, TCMSP database and the latest research literatures. The chemical molecular library was established after class prediction and screening for medicinal and metabolic properties. Then, five kinds of vasodilatory and vasoconstrictory related GPCR crystal structure including 5-HT receptors (5-HT1AR, 5-HT1BR), AT1R, β2-AR, hUTR and ETB were retrieved from RCSB Protein Data Bank database or constructed by homology modeling of Discovery Studio 4.1 built-in modeling tools. After virtual screening by Libdock molecular docking, the highest rated 50 compounds of each target were collected and analyzed. The collected data were further used to construct and analyze the network by Cytoscape 3.4.0. The results showed that most of the chemical composition effects were associated with different vasodilatory and vasoconstrictory related GPCR targets, while a few effective components could be applied to multiple GPCR targets at the same time, therefore forming synergies and vasorelaxant effects of XXMD.

Endo-lysosomal sorting of G-protein-coupled receptors by ubiquitin: Diverse pathways for G-protein-coupled receptor destruction and beyond.

Ubiquitin is covalently attached to substrate proteins in the form of a single ubiquitin moiety or polyubiquitin chains and has been generally linked to protein degradation, however, distinct types of ubiquitin linkages are also used to control other critical cellular processes like cell signaling. Over forty mammalian G protein-coupled receptors (GPCRs) have been reported to be ubiquitinated, but despite the diverse and rich complexity of GPCR signaling, ubiquitin has been largely ascribed to receptor degradation. Indeed, GPCR ubiquitination targets the receptors for degradation by lysosome, which is mediated by the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery, and the proteasome. This has led to the view that ubiquitin and ESCRTs primarily function as the signal to target GPCRs for destruction. Contrary to this conventional view, studies indicate that ubiquitination of certain GPCRs and canonical ubiquitin-binding ESCRTs are not required for receptor degradation and revealed that diverse and complex pathways exist to regulate endo-lysosomal sorting of GPCRs. In other studies, GPCR ubiquitination has been shown to drive signaling and not receptor degradation and further revealed novel insight into the mechanisms by which GPCRs trigger the activity of the ubiquitination machinery. Here, we discuss the diverse pathways by which ubiquitin controls GPCR endo-lysosomal sorting and beyond.

3-Iodothyronamine Activates a Set of Membrane Proteins in Murine Hypothalamic Cell Lines.

3-Iodothyronamine (3-T1AM) is an endogenous thyroid hormone metabolite. The profound pharmacological effects of 3-T1AM on energy metabolism and thermal homeostasis have raised interest to elucidate its signaling properties in tissues that pertain to metabolic regulation and thermogenesis. Previous studies identified G protein-coupled receptors (GPCRs) and transient receptor potential channels (TRPs) as targets of 3-T1AM in different cell types. These two superfamilies of membrane proteins are largely expressed in tissue which influences energy balance and metabolism. As the first indication that 3-T1AM virtually modulates the function of the neurons in hypothalamus, we observed that intraperitoneal administration of 50 mg/kg bodyweight of 3-T1AM significantly increased the c-FOS activation in the paraventricular nucleus (PVN) of C57BL/6 mice. To elucidate the underlying mechanism behind this 3-T1AM-induced signalosome, we used three different murine hypothalamic cell lines, which are all known to express PVN markers, GT1-7, mHypoE-N39 (N39) and mHypoE-N41 (N41). Various aminergic GPCRs, which are the known targets of 3-T1AM, as well as numerous members of TRP channel superfamily, are expressed in these cell lines. Effects of 3-T1AM on activation of GPCRs were tested for the two major signaling pathways, the action of Gαs/adenylyl cyclase and Gi/o. Here, we demonstrated that this thyroid hormone metabolite has no significant effect on Gi/o signaling and only a minor effect on the Gαs/adenylyl cyclase pathway, despite the expression of known GPCR targets of 3-T1AM. Next, to test for other potential mechanisms involved in 3-T1AM-induced c-FOS activation in PVN, we evaluated the effect of 3-T1AM on the intracellular Ca2+ concentration and whole-cell currents. The fluorescence-optic measurements showed a significant increase of intracellular Ca2+ concentration in the three cell lines in the presence of 10 μM 3-T1AM. Furthermore, this thyroid hormone metabolite led to an increase of whole-cell currents in N41 cells. Interestingly, the TRPM8 selective inhibitor (10 μM AMTB) reduced the 3-T1AM stimulatory effects on cytosolic Ca2+ and whole-cell currents. Our results suggest that the profound pharmacological effects of 3-T1AM on selected brain nuclei of murine hypothalamus, which are known to be involved in energy metabolism and thermoregulation, might be partially attributable to TRP channel activation in hypothalamic cells.

Label-free impedance-based whole cell assay to study GPCR pharmacology.

The superfamily of G protein-coupled receptors (GPCRs) represents the largest group of cell surface receptors in the human body. It is estimated that around 40% of the drugs currently on the market target GPCRs. As only a very small number of GPCRs is targeted by these marketed drugs, the potential of GPCRs as novel drug targets remains enormous. As opposed to conventional in vitro assays, label-free cellular assays using a biosensor provide new opportunities for studying GPCRs. Integrated receptor-mediated responses are measured in real-time rather than a single downstream signaling pathway, without the need for the use of any label (e.g., fluorescent or radioactive). Here, we describe a protocol to study GPCR pharmacology using the label-free whole cell impedance-based biosensor system xCELLigence. This assay allows quantification of compound-induced GPCR-mediated responses in real-time. Finally, we have also discussed the analysis and interpretation of the results obtained with this assay using the mGlu2 receptor as a model system.

A luciferase complementation assay system using transferable mouse artificial chromosomes to monitor protein\u2013protein interactions mediated by G protein-coupled receptors

G protein-coupled receptors (GPCRs) are seven-transmembrane domain receptors that interact with the β-arrestin family, particularly β-arrestin 1 (ARRB1). GPCRs interact with 33% of small molecule drugs. Ligand screening is promising for drug discovery concerning GPCR-related diseases. Luciferase complementation assay (LCA) enables detection of protein–protein complementation via bioluminescence following complementation of N- and C-terminal luciferase fragments (NEluc and CEluc) fused to target proteins, but it is necessary to co-express the two genes. Here, we developed LCAs with mouse artificial chromosomes (MACs) that have unique characteristics such as stable maintenance and a substantial insert-carrying capacity. First, an NEluc-ARRB1 was inserted into MAC4 by Cre-loxP recombination in CHO cells, named ARRB1-MAC4. Second, a parathyroid hormone receptor 2 (PTHR2)-CEluc or prostaglandin EP4 receptor (hEP4)-CEluc were inserted into ARRB1-MAC4, named ARRB1-PTHR2-MAC4 and ARRB1-hEP4-MAC4, respectively, via the sequential integration of multiple vectors (SIM) system. Each MAC was transferred into HEK293 cells by microcell-mediated chromosome transfer (MMCT). LCAs using the established HEK293 cell lines resulted in 35,000 photon counts upon somatostatin stimulation for ARRB1-MAC4 with transient transfection of the somatostatin receptor 2 (SSTR2) expression vector, 1800 photon counts upon parathyroid hormone stimulation for ARRB1-PTHR2-MAC4, and 35,000 photon counts upon prostaglandin E2 stimulation for ARRB1-hEP4-MAC4. These MACs were maintained independently from host chromosomes in CHO and HEK293 cells. HEK293 cells containing ARRB1-PTHR2-MAC4 showed a stable reaction for long-term. Thus, the combination of gene loading by the SIM system into a MAC and an LCA targeting GPCRs provides a novel and useful platform to discover drugs for GPCR-related diseases.