scholarly journals The habenular G-protein–coupled receptor 151 regulates synaptic plasticity and nicotine intake

2020 ◽  
Vol 117 (10) ◽  
pp. 5502-5509 ◽  
Author(s):  
Beatriz Antolin-Fontes ◽  
Kun Li ◽  
Jessica L. Ables ◽  
Michael H. Riad ◽  
Andreas Görlich ◽  
...  
Keyword(s):  
G Protein ◽  
Nicotinic Acetylcholine Receptors ◽  
Brain Area ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Nicotine Addiction ◽  
G Protein Coupled Receptor ◽  
Inhibitory Protein ◽  
Nicotine Intake ◽  
G Protein Coupled

The habenula, an ancient small brain area in the epithalamus, densely expresses nicotinic acetylcholine receptors and is critical for nicotine intake and aversion. As such, identification of strategies to manipulate habenular activity may yield approaches to treat nicotine addiction. Here we show that GPR151, an orphan G-protein–coupled receptor (GPCR) highly enriched in the habenula of humans and rodents, is expressed at presynaptic membranes and synaptic vesicles and associates with synaptic components controlling vesicle release and ion transport. Deletion of Gpr151 inhibits evoked neurotransmission but enhances spontaneous miniature synaptic currents and eliminates short-term plasticity induced by nicotine. We find that GPR151 couples to the G-alpha inhibitory protein Gαo1 to reduce cyclic adenosine monophosphate (cAMP) levels in mice and in GPR151-expressing cell lines that are amenable to ligand screens. Gpr151– knockout (KO) mice show diminished behavioral responses to nicotine and self-administer greater quantities of the drug, phenotypes rescued by viral reexpression of Gpr151 in the habenula. These data identify GPR151 as a critical modulator of habenular function that controls nicotine addiction vulnerability.

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

2000 ◽  
Vol 5 (4) ◽  
pp. 239-247 ◽  
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.

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

2006 ◽  
Vol 11 (4) ◽  
pp. 351-358 ◽  
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.

scholarly journals Identification of ligand-specific G protein-coupled receptor states and prediction of downstream efficacy via data-driven modeling

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Oliver Fleetwood ◽  
Jens Carlsson ◽  
Lucie Delemotte
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
G Protein ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Conformational Ensemble ◽  
G Protein Coupled Receptor ◽  
Dynamics Simulations ◽  
Data Driven Modeling ◽  
G Protein Coupled

Ligand binding stabilizes different G protein-coupled receptor states via a complex allosteric process that is not completely understood. Here, we have derived free energy landscapes describing activation of the β2 adrenergic receptor bound to ligands with different efficacy profiles using enhanced sampling molecular dynamics simulations. These reveal shifts toward active-like states at the Gprotein-binding site for receptors bound to partial and full agonists, and that the ligands modulate the conformational ensemble of the receptor by tuning protein microswitches. We indeed find an excellent correlation between the conformation of the microswitches close to the ligand binding site and in the transmembrane region and experimentally reported cyclic adenosine monophosphate signaling responses. Dimensionality reduction further reveals the similarity between the unique conformational states induced by different ligands, and examining the output of classifiers highlights two distant hotspots governing agonism on transmembrane helices 5 and 7.

scholarly journals Conserved salt-bridge competition triggered by phosphorylation regulates the protein interactome

2017 ◽  
Vol 114 (51) ◽  
pp. 13453-13458 ◽  
Author(s):  
John J. Skinner ◽  
Sheng Wang ◽  
Jiyoung Lee ◽  
Colin Ong ◽  
Ruth Sommese ◽  
...  
Keyword(s):  
G Protein ◽  
Protein Interactions ◽  
Salt Bridge ◽  
G Protein Coupled Receptor ◽  
Inhibitory Protein ◽  
Raf Kinase ◽  
Protein Interfaces ◽  
Partial Unfolding ◽  
Direct Recognition ◽  
G Protein Coupled

Phosphorylation is a major regulator of protein interactions; however, the mechanisms by which regulation occurs are not well understood. Here we identify a salt-bridge competition or “theft” mechanism that enables a phospho-triggered swap of protein partners by Raf Kinase Inhibitory Protein (RKIP). RKIP transitions from inhibiting Raf-1 to inhibiting G-protein–coupled receptor kinase 2 upon phosphorylation, thereby bridging MAP kinase and G-Protein–Coupled Receptor signaling. NMR and crystallography indicate that a phosphoserine, but not a phosphomimetic, competes for a lysine from a preexisting salt bridge, initiating a partial unfolding event and promoting new protein interactions. Structural elements underlying the theft occurred early in evolution and are found in 10% of homo-oligomers and 30% of hetero-oligomers including Bax, Troponin C, and Early Endosome Antigen 1. In contrast to a direct recognition of phosphorylated residues by binding partners, the salt-bridge theft mechanism represents a facile strategy for promoting or disrupting protein interactions using solvent-accessible residues, and it can provide additional specificity at protein interfaces through local unfolding or conformational change.

scholarly journals The Habenular Receptor GPR151 Regulates Addiction Vulnerability Across Drug Classes

2019 ◽  
Author(s):  
Beatriz Antolin-Fontes ◽  
Kun Li ◽  
Jessica L. Ables ◽  
Michael H. Riad ◽  
Andreas Görlich ◽  
...  
Keyword(s):  
G Protein ◽  
Cannabinoid Receptors ◽  
Behavioral Responses ◽  
G Protein Coupled Receptor ◽  
Inhibitory Protein ◽  
Vesicle Release ◽  
Release Probability ◽  
Drug Classes ◽  
G Protein Coupled ◽  
Camp Levels

SUMMARYThe habenula controls the addictive properties of nicotine but also densely expresses opioid and cannabinoid receptors. As such, identification of strategies to manipulate habenular activity may yield new approaches to treat substance use disorders. Here we show that GPR151, an orphan G protein-coupled receptor (GPCR) highly enriched in the habenula of humans and rodents plays a critical role in regulating habenular function and behavioral responses to addictive drugs. We show that GPR151 is expressed on axonal and presynaptic membranes and synaptic vesicles, and regulates synaptic fidelity and plasticity. We find that GPR151 associates with synaptic components controlling vesicle release and ion transport and couples to the G-alpha inhibitory protein Gαo1to reduce cAMP levels. Stable cell lines expressing GPR151 confirm that it signals via Gi/o and are amenable to ligand screens.Gpr151null mice show diminished behavioral responses to nicotine, and self-administer greater quantities of the drug, phenotypes rescued by viral re-expression ofGpr151in the habenula.Gpr151null mice are also insensitive to the behavioral actions of morphine and cannabinoids. These data identify GPR151 as a critical modulator of habenular function that controls addiction vulnerability across different drug classes.HighlightsHabenula neurons are enriched in nicotinic, opioid, cannabinoid and GPR151 receptorsGPR151 modulates synaptic fidelity and release probability at habenular terminals.Habenular GPR151 plays a role in drug abuse and food intake/weight controlGPR151 couples to the G-alpha inhibitory protein Gαo1to reduce cAMP levels.eTOC BlurbAntolin-Fontes at al. identify a G protein-coupled receptor, GPR151, which is highly enriched in human habenular neurons. These neurons are primarily enriched with nicotinic, opioid and cannabinoid receptors. We find that GPR151 modulates habenular synaptic vesicle release probability and behavioral responses to these drugs of abuse.

scholarly journals GPRC5A: An Emerging Biomarker in Human Cancer

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
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.

scholarly journals A Novel Cyclic Adenosine Monophosphate–Responsive Luciferase Reporter Incorporating a Nonpalindromic Cyclic Adenosine Monophosphate Response Element Provides Optimal Performance for Use in G Protein–Coupled Receptor Drug Discovery Efforts

2007 ◽  
Vol 12 (5) ◽  
pp. 740-746 ◽  
Author(s):  
Oleg G. Chepurny ◽  
George G. Holz
Keyword(s):  
G Protein ◽  
Dynamic Range ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Firefly Luciferase ◽  
Optimal Performance ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Response Element ◽  
G Protein Coupled

The authors report the characterization of a novel cyclic adenosine monophosphate (cAMP)—responsive luciferase (Luc) reporter that exhibits optimal performance in high-throughput screens of agonist binding at G protein—coupled receptors (GPCRs). This reporter (RIP1-CRE-Luc) incorporates a nonpalindromic cAMP response element (CRE) originally identified within the 5′ promoter of the rat insulin 1 gene (RIP1). When multimerized and fused to the coding sequence of firefly luciferase, the CRE of RIP1 allows for the efficient activation of luciferase expression by cAMP-elevating agents or by cAMP itself. Of primary importance is the demonstration that RIP1-CRE-Luc does not exhibit the relatively high levels of basal luciferase activity inherent to reporters incorporating the palindromic CRE first identified in the somatostatin gene promoter. Furthermore, studies of HEK cells expressing class II GPCRs for the cAMP-elevating hormones GLP-1, GIP, and glucagon demonstrate that RIP1-CRE-Luc affords a much wider dynamic range of activation upon exposure to agonist. Such properties of RIP1-CRE-Luc indicate its usefulness as a new and powerful tool for the identification of small-molecule compounds with receptor-stimulating actions or for the identification of constitutively active orphan receptors with cAMP-signaling properties. ( Journal of Biomolecular Screening 2007:740-746)

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

2005 ◽  
Vol 10 (7) ◽  
pp. 730-737 ◽  
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.

scholarly journals Extensive crosstalk of G protein-coupled receptors with the Hedgehog signalling pathway

Development ◽  
2021 ◽  
pp. dev.189258
Author(s):  
Farah Saad ◽  
David R. Hipfner
Keyword(s):  
G Protein ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
G Protein Coupled Receptors ◽  
Signalling Pathway ◽  
Protein Activity ◽  
Cellular Sensitivity ◽  
Hedgehog Signalling Pathway ◽  
G Protein Coupled ◽  
Camp Levels

Hedgehog (Hh) ligands orchestrate tissue patterning and growth by acting as morphogens, dictating different cellular responses depending on ligand concentration. Cellular sensitivity to Hh ligands is influenced by heterotrimeric G protein activity, which controls production of the second messenger 3',5'-cyclic adenosine monophosphate (cAMP). cAMP in turn activates Protein kinase A (PKA), which functions as an inhibitor and (uniquely in Drosophila) an activator of Hh signalling. A few mammalian Gαi- and Gαs-coupled G protein-coupled receptors (GPCRs) have been shown to influence Sonic Hh (Shh) responses in this way. To determine if this is a more general phenomenon, we carried out an RNAi screen targeting GPCRs in Drosophila. RNAi-mediated depletion of more than 40% of GPCRs tested either decreased or increased Hh responsiveness in the developing Drosophila wing, closely matching the effects of Gαs and Gαi depletion, respectively. Genetic analysis indicated that the orphan GPCR Mthl5 lowers cAMP levels to attenuate Hh responsiveness. Our results identify Mthl5 as a new Hh signalling pathway modulator in Drosophila and suggest that many GPCRs may crosstalk with the Hh pathway in mammals.

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