Nomad Biosensors: A New Multiplexed Technology for the Screening of GPCR Ligands

Nomad Technology (Innoprot [Innovative Technologies in Biological Systems], Derio, Spain), a novel tool for multiplexing high-throughput cell-based G protein–coupled receptor (GPCR) assays, is described in this work. This new technology comprises a family of fluorescent biosensors called Nomad Biosensors that allow for the measurement of responses mediated by G proteins through their interactions with second-messenger transduction proteins. GPCRs are one of the largest protein families of receptors in eukaryotes, and their signaling mediates important physiological processes within cells. Thus, GPCRs are associated with a wide variety of diseases, and considered major targets in therapeutic research. Nomad constitutes a novel tool for unraveling the mechanism of GPCR signal transduction by simultaneously tracing different pathways. GPCR activation changes the structural folding of the biosensor and promotes its vesicularization, as well as an increase in the fluorescence intensity. Based on this technology, the MPXNomad cellular model was developed to discriminate between the Ca2+-mediated pathway and the cyclic adenosine monophosphate (cAMP)–mediated pathway. To validate this model, endothelin receptor B (ETBR) was coexpressed into the MPXNomad cell line and assessed with a specific agonist, an antagonist, and a chemical library of compounds. Nomad Technology optimizes the identification of novel GPCR ligands and enables the testing of large numbers of compounds.

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GPCR Screening via ERK 1/2: A Novel Platform for Screening G Protein–Coupled Receptors

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057105277968 ◽

2005 ◽

Vol 10 (7) ◽

pp. 730-737 ◽

Cited By ~ 46

Author(s):

Ronald I. W. Osmond ◽

Antony Sheehan ◽

Romana Borowicz ◽

Emma Barnett ◽

Georgina Harvey ◽

...

Keyword(s):

G Protein ◽

High Throughput ◽

Measurement Techniques ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

G Protein Coupled Receptors ◽

Drug Candidates ◽

Gpcr Activation ◽

Intracellular Ca ◽

G Protein Coupled

Discovery of novel agonists and antagonists for G protein–coupled receptors (GPCRs) relies heavily on cell-based assays because determination of functional consequences of receptor engagement is often desirable. Currently, there are several key parameters measured to achieve this, including mobilization of intracellular Ca2+ and formation of cyclic adenosine monophosphate or inositol triphosphate. However, no single assay platform is suitable for all situations, and all of the assays have limitations. The authors have developed a new high-throughput homogeneous assay platform for GPCR discovery as an alternative to current assays, which employs detection of phosphorylation of the key signaling molecule p42/44 MAP kinase (ERK 1/2). The authors show that ERK 1/2 is consistently activated in cells stimulated by Gq-coupled GPCRs and provides a new high-throughput platform for screening GPCR drug candidates. The activation of ERK 1/2 in Gq-coupled GPCR systems generates comparable pharmacological data for receptor agonist and antagonist data obtained by other GPCR activation measurement techniques.

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Mapping the Heart

Microscopy Today ◽

10.1017/s155192951000043x ◽

2010 ◽

Vol 18 (4) ◽

pp. 6-8

Author(s):

Stephen W. Carmichael

Keyword(s):

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

G Protein Coupled Receptors ◽

Receptor Subtypes ◽

Cardiac Muscle Cells ◽

Guanine Nucleotide Binding Protein ◽

The Common ◽

Guanine Nucleotide Binding ◽

First Time ◽

G Protein Coupled

Some of the receptors on the surface of cardiac muscle cells (cardiomyocytes) mediate the response of these cells to catecholamines by causing the production of the common second messenger cyclic adenosine monophosphate (cAMP). An example of such receptors are the β1- and β2-adrenergic receptors (βARs) that are heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors. Selective stimulation of these two receptor subtypes leads to distinct physiological and pathophysiological responses, but their precise location on the surface of cardiomyocytes has not been correlated with these responses. In an ingenious combination of techniques, Viacheslav Nikolaev, Alexey Moshkov, Alexander Lyon, Michele Miragoli, Pavel Novak, Helen Paur, Martin Lohse, Yuri Korchev, Sian Harding, and Julia Gorelik have mapped the function of these receptors for the first time.

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Characterization of the Signaling Modalities of Prostaglandin E2 Receptors EP2 and EP4 Reveals Crosstalk and a Role for Microtubules

Frontiers in Immunology ◽

10.3389/fimmu.2020.613286 ◽

2021 ◽

Vol 11 ◽

Author(s):

Ward Vleeshouwers ◽

Koen van den Dries ◽

Sandra de Keijzer ◽

Ben Joosten ◽

Diane S. Lidke ◽

...

Keyword(s):

Prostaglandin E2 ◽

Quantitative Imaging ◽

Myeloid Cells ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Lipid Mediator ◽

Camp Production ◽

Microtubule Network ◽

Immune Modulators ◽

G Protein Coupled

Prostaglandin E2 (PGE2) is a lipid mediator that modulates the function of myeloid immune cells such as macrophages and dendritic cells (DCs) through the activation of the G protein-coupled receptors EP2 and EP4. While both EP2 and EP4 signaling leads to an elevation of intracellular cyclic adenosine monophosphate (cAMP) levels through the stimulating Gαs protein, EP4 also couples to the inhibitory Gαi protein to decrease the production of cAMP. The receptor-specific contributions to downstream immune modulatory functions are still poorly defined. Here, we employed quantitative imaging methods to characterize the early EP2 and EP4 signaling events in myeloid cells and their contribution to the dissolution of adhesion structures called podosomes, which is a first and essential step in DC maturation. We first show that podosome loss in DCs is primarily mediated by EP4. Next, we demonstrate that EP2 and EP4 signaling leads to distinct cAMP production profiles, with EP4 inducing a transient cAMP response and EP2 inducing a sustained cAMP response only at high PGE2 levels. We further find that simultaneous EP2 and EP4 stimulation attenuates cAMP production, suggesting a reciprocal control of EP2 and EP4 signaling. Finally, we demonstrate that efficient signaling of both EP2 and EP4 relies on an intact microtubule network. Together, these results enhance our understanding of early EP2 and EP4 signaling in myeloid cells. Considering that modulation of PGE2 signaling is regarded as an important therapeutic possibility in anti-tumor immunotherapy, our findings may facilitate the development of efficient and specific immune modulators of PGE2 receptors.

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Identification and characterization of an atypical Gαs-biased β2AR agonist that fails to evoke airway smooth muscle cell tachyphylaxis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2026668118 ◽

2021 ◽

Vol 118 (49) ◽

pp. e2026668118

Author(s):

Donghwa Kim ◽

Alina Tokmakova ◽

Lauren K. Lujan ◽

Hannah R. Strzelinski ◽

Nicholas Kim ◽

...

Keyword(s):

Smooth Muscle ◽

Airway Smooth Muscle ◽

Chemical Space ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Airway Smooth Muscle Cell ◽

Functional Studies ◽

G Protein Coupled ◽

Identification And Characterization

G protein–coupled receptors display multifunctional signaling, offering the potential for agonist structures to promote conformational selectivity for biased outputs. For β2-adrenergic receptors (β2AR), unbiased agonists stabilize conformation(s) that evoke coupling to Gαs (cyclic adenosine monophosphate [cAMP] production/human airway smooth muscle [HASM] cell relaxation) and β-arrestin engagement, the latter acting to quench Gαs signaling, contributing to receptor desensitization/tachyphylaxis. We screened a 40-million-compound scaffold ranking library, revealing unanticipated agonists with dihydroimidazolyl-butyl-cyclic urea scaffolds. The S-stereoisomer of compound C1 shows no detectable β-arrestin engagement/signaling by four methods. However, C1-S retained Gαs signaling—a divergence of the outputs favorable for treating asthma. Functional studies with two models confirmed the biasing: β2AR-mediated cAMP signaling underwent desensitization to the unbiased agonist albuterol but not to C1-S, and desensitization of HASM cell relaxation was observed with albuterol but not with C1-S. These HASM results indicate biologically pertinent biasing of C1-S, in the context of the relevant physiologic response, in the human cell type of interest. Thus, C1-S was apparently strongly biased away from β-arrestin, in contrast to albuterol and C5-S. C1-S structural modeling and simulations revealed binding differences compared with unbiased epinephrine at transmembrane (TM) segments 3,5,6,7 and ECL2. C1-S (R2 = cyclohexane) was repositioned in the pocket such that it lost a TM6 interaction and gained a TM7 interaction compared with the analogous unbiased C5-S (R2 = benzene group), which appears to contribute to C1-S biasing away from β-arrestin. Thus, an agnostic large chemical-space library identified agonists with receptor interactions that resulted in relevant signal splitting of β2AR actions favorable for treating obstructive lung disease.

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A Novel High Throughput Chemiluminescent Assay for the Measurement of Cellular Cyclic Adenosine Monophosphate Levels

CrossRef Listing of Deleted DOIs ◽

10.1177/108705710000500406 ◽

2000 ◽

Vol 5 (4) ◽

pp. 239-247 ◽

Cited By ~ 15

Author(s):

Anthony C. Chiulli ◽

Karen Trompeter ◽

Michelle Palmer

Keyword(s):

G Protein ◽

High Throughput ◽

High Throughput Screening ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Receptor Activation ◽

G Protein Coupled Receptor ◽

Wide Range ◽

G Protein Coupled ◽

Camp Levels

The second messenger 3′, 5′-cyclic AMP (cAMP) is a highly regulated molecule that is governed by G protein-coupled receptor activation and other cellular processes. Measurement of cAMP levels in cells is widely used as an indicator of receptor function in drug discovery applications. We have developed a nonradioactive ELISA for the accurate quantitation of cAMP levels produced in cell-based assays. This novel competitive assay utilizes chemiluminescent detection that affords both a sensitivity and a dynamic assay range that have not been previously reported with any other assay methodologies. The assay has been automated in 96- and 384-well formats, providing assay data that are equivalent to, if not better than, data generated by hand. This report demonstrates the application of this novel assay technology to the functional analysis of a specific G protein-coupled receptor, neuropeptide receptor Y1, on SK-N-MC cells. Our data indicate the feasibility of utilizing this assay methodology for monitoring cAMP levels in a wide range of functional cell-based assays for high throughput screening.

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GPRC5A: An Emerging Biomarker in Human Cancer

BioMed Research International ◽

10.1155/2018/1823726 ◽

2018 ◽

Vol 2018 ◽

pp. 1-11 ◽

Cited By ~ 3

Author(s):

Xiaoxia Jiang ◽

Xin Xu ◽

Mengjie Wu ◽

Zhonghai Guan ◽

Xingyun Su ◽

...

Keyword(s):

Signaling Pathways ◽

G Protein ◽

Molecular Mechanisms ◽

Lung Tumor ◽

Human Cancer ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Aberrant Expression ◽

Human Cancers ◽

G Protein Coupled

Aberrant expression of G protein-coupled receptors (GPCRs) is frequently associated with tumorigenesis. G Protein-coupled receptor class C group 5 member A (GPRC5A) is a member of the GPCR superfamily, is expressed preferentially in lung tissues, and is regulated by various entities at multiple levels. GPRC5A exerts a tumor suppressive role in lung cancer and GPRC5A deletion promotes lung tumor initiation and progression. Recent advances have highlighted that GPRC5A dysregulation is found in various human cancers and is related to many tumor-associated signaling pathways, including the cyclic adenosine monophosphate (cAMP), nuclear factor (NF)-κB, signal transducer and activator of transcription (STAT) 3, and focal adhesion kinase (FAK)/Src signaling. This review aimed to summarize our updated view on the biology and regulation of GPRC5A, its expression in human cancers, and the linked signaling pathways. A better comprehension of the underlying cellular and molecular mechanisms of GPRC5A will provide novel insights into its potential diagnostic and therapeutic value.

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Forskolin Inhibits Lipopolysaccharide-Induced Modulation of MCP-1 and GPR120 in 3T3-L1 Adipocytes through an Inhibition of NFκB

Mediators of Inflammation ◽

10.1155/2016/1431789 ◽

2016 ◽

Vol 2016 ◽

pp. 1-11 ◽

Cited By ~ 2

Author(s):

Jeanne Durendale Chiadak ◽

Tatjana Arsenijevic ◽

Kevin Verstrepen ◽

Françoise Gregoire ◽

Nargis Bolaky ◽

...

Keyword(s):

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Mrna Levels ◽

Nuclear Factor ◽

Obese Adult ◽

Toll Like Receptor ◽

Toll Like Receptor 4 ◽

Adult Men ◽

Chemotactic Protein ◽

G Protein Coupled

In an obese state, Toll-like receptor-4 (TLR-4) upregulates proinflammatory adipokines secretion including monocyte chemotactic protein-1 (MCP-1) in adipose tissue. In contrast, G-protein coupled receptor 120 (GPR120) mediates antiobesity effects. The aim of this study was to determine the signaling pathway by which Forskolin (FK), a cyclic adenosine monophosphate- (cAMP-) promoting agent causing positive changes in body composition in overweight and obese adult men, affects MCP-1 and GPR120 expression during an inflammatory response induced by lipopolysaccharide (LPS) in adipocytes, such as in an obese state. 3T3-L1 cells differentiated into adipocytes (DC) were stimulated with LPS in the absence or presence of FK and inhibitors of TLR-4 and inhibitor of kappa B (IκBα). In DC, LPS increased MCP-1, TLR-4, and nuclear factor-κB1 (NFκB1) mRNA levels, whereas it decreased GPR120 mRNA levels. In DC, FK inhibited the LPS-induced increase in MCP-1, TLR-4, and NFκB1 mRNA levels and the LPS-induced decrease in GPR120 mRNA. BAY11-7082 and CLI-095 abolished these LPS-induced effects. In conclusion, FK inhibits LPS-induced increase in MCP-1 mRNA levels and decrease in GPR120 mRNA levels in adipocytes and may be a potential treatment for inflammation in obesity. Furthermore, TLR-4-induced activation of NFκB may be involved in the LPS-induced regulation of these genes.

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A Novel Cell-Based Assay for G-Protein-Coupled Receptor-Mediated Cyclic Adenosine Monophosphate Response Element Binding Protein Phosphorylation

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057106286608 ◽

2006 ◽

Vol 11 (4) ◽

pp. 351-358 ◽

Cited By ~ 18

Author(s):

Julie V. Selkirk ◽

Lisa M. Nottebaum ◽

Ian C. Ford ◽

Mark Santos ◽

Siobhan Malany ◽

...

Keyword(s):

G Protein ◽

Binding Protein ◽

Expression System ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

G Protein Coupled Receptor ◽

Response Element ◽

Creb Phosphorylation ◽

Element Binding Protein ◽

G Protein Coupled

Currently, the most popular means of assessing functional activity of Gs/olf-coupled receptors is via the measurement of intracellular cyclic adenosine monophosphate (cAMP) accumulation. An additional readout is the downstream phosphorylation of cAMP response element binding protein (CREB), which gives an indication of gene transcription, the ultimate response of many G-protein-coupled receptor (GPCR) signals. Current methods of quantifying CREB phosphorylation are low throughput, and so we have designed a novel higher throughput method using the Odyssey™ infrared imaging system. Functional potencies of both agonists and antagonists correlate well with radioligand binding affinities determined using examples of both an endogenous (adenosine2A receptor in PC-12 cells) and a heterologous (human melanocortin 4 receptor in HEK-293 cells) expression system. For example, the antagonist ZM241385 demonstrates 0.23 ± 0.03 nM affinity for the A2A receptor and has a functional potency of 0.26 ± 0.04 nM determined using cAMP and 0.15 ± 0.06 nM using CREB phosphorylation. These data demonstrate that this novel approach for the measurement of CREB phosphorylation is a useful tool for the assessment of GPCR activity in whole cells and is more amenable to the throughput required for the purposes of drug discovery.

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FluoSTEPs: Fluorescent biosensors for monitoring compartmentalized signaling within endogenous microdomains

Science Advances ◽

10.1126/sciadv.abe4091 ◽

2021 ◽

Vol 7 (21) ◽

pp. eabe4091

Author(s):

Brian Tenner ◽

Jason Z. Zhang ◽

Yonghoon Kwon ◽

Veronica Pessino ◽

Siyu Feng ◽

...

Keyword(s):

Intracellular Signaling ◽

Fluorescent Protein ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Regulatory Elements ◽

Live Cells ◽

Fluorescent Biosensors ◽

Signaling Events

Growing evidence suggests that many essential intracellular signaling events are compartmentalized within kinetically distinct microdomains in cells. Genetically encoded fluorescent biosensors are powerful tools to dissect compartmentalized signaling, but current approaches to probe these microdomains typically rely on biosensor fusion and overexpression of critical regulatory elements. Here, we present a novel class of biosensors named FluoSTEPs (fluorescent sensors targeted to endogenous proteins) that combine self-complementing split green fluorescent protein, CRISPR-mediated knock-in, and fluorescence resonance energy transfer biosensor technology to probe compartmentalized signaling dynamics in situ. We designed FluoSTEPs for simultaneously highlighting endogenous microdomains and reporting domain-specific, real-time signaling events including kinase activities, guanosine triphosphatase activation, and second messenger dynamics in live cells. A FluoSTEP for 3′,5′-cyclic adenosine monophosphate (cAMP) revealed distinct cAMP dynamics within clathrin microdomains in response to stimulation of G protein–coupled receptors, showcasing the utility of FluoSTEPs in probing spatiotemporal regulation within endogenous signaling architectures.

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Characterization of the signalling modalities of prostaglandin E2 receptors EP2 and EP4 reveals crosstalk and a role for microtubules

10.1101/2020.10.06.328351 ◽

2020 ◽

Author(s):

Ward Vleeshouwers ◽

Koen van den Dries ◽

Sandra de Keijzer ◽

Ben Joosten ◽

Diane S. Lidke ◽

...

Keyword(s):

Prostaglandin E2 ◽

Quantitative Imaging ◽

Myeloid Cells ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Lipid Mediator ◽

Camp Production ◽

Microtubule Network ◽

Immune Modulators ◽

G Protein Coupled

AbstractProstaglandin E2 (PGE2) is a lipid mediator that modulates the function of myeloid immune cells such as macrophages and dendritic cells (DCs) through the activation of the G protein-coupled receptors EP2 and EP4. While both EP2 and EP4 signalling leads to an elevation of intracellular cyclic adenosine monophosphate (cAMP) levels through the stimulating Gαs protein, EP4 also couples to the inhibitory Gαi protein to decrease the production of cAMP. The receptor-specific contributions to downstream immune modulatory functions are still poorly defined. Here, we employed quantitative imaging methods to characterize the early EP2 and EP4 signalling events in myeloid cells and their contribution to the dissolution of adhesion structures called podosomes, which is a first and essential step in DC maturation. We first show that podosome loss in DCs is primarily mediated by EP4. Next, we demonstrate that EP2 and EP4 signalling leads to distinct cAMP production profiles, with EP4 inducing a transient cAMP response and EP2 inducing a sustained cAMP response only at high PGE2 levels. We further find that simultaneous EP2 and EP4 stimulation attenuates cAMP production, suggesting a reciprocal control of EP2 and EP4 signaling. Finally, we demonstrate that efficient signaling of both EP2 and EP4 relies on an intact microtubule network. Together, these results enhance our understanding of early EP2 and EP4 signalling in myeloid cells. Considering that modulation of PGE2 signalling is regarded as an important therapeutic possibility in anti-tumour immunotherapy, our findings may facilitate the development of efficient and specific immune modulators of PGE2 receptors.

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