scholarly journals Determination of the melanocortin-4 receptor structure identifies Ca2+ as a cofactor for ligand binding

Science ◽  
2020 ◽  
Vol 368 (6489) ◽  
pp. 428-433 ◽  
Author(s):  
Jing Yu ◽  
Luis E. Gimenez ◽  
Ciria C. Hernandez ◽  
Yiran Wu ◽  
Ariel H. Wein ◽  
...  
Keyword(s):  
G Protein ◽  
Energy Homeostasis ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Peptide Ligand ◽  
Melanocyte Stimulating Hormone ◽  
Melanocortin 4 Receptor ◽  
Syndromic Obesity ◽  
G Protein Coupled

The melanocortin-4 receptor (MC4R) is involved in energy homeostasis and is an important drug target for syndromic obesity. We report the structure of the antagonist SHU9119-bound human MC4R at 2.8-angstrom resolution. Ca2+ is identified as a cofactor that is complexed with residues from both the receptor and peptide ligand. Extracellular Ca2+ increases the affinity and potency of the endogenous agonist α-melanocyte–stimulating hormone at the MC4R by 37- and 600-fold, respectively. The ability of the MC4R crystallized construct to couple to ion channel Kir7.1, while lacking cyclic adenosine monophosphate stimulation, highlights a heterotrimeric GTP-binding protein (G protein)–independent mechanism for this signaling modality. MC4R is revealed as a structurally divergent G protein–coupled receptor (GPCR), with more similarity to lipidic GPCRs than to the homologous peptidic GPCRs.

scholarly journals Differential Signaling Profiles of MC4R Mutations with Three Different Ligands

2020 ◽  
Vol 21 (4) ◽  
pp. 1224 ◽  
Author(s):  
Sarah Paisdzior ◽  
Ioanna Maria Dimitriou ◽  
Paul Curtis Schöpe ◽  
Paolo Annibale ◽  
Patrick Scheerer ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
G Protein ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Loss Of Function ◽  
Melanocyte Stimulating Hormone ◽  
Melanocortin 4 Receptor ◽  
Erk Phosphorylation ◽  
Extracellular Regulated Kinase ◽  
Peptide Agonist

The melanocortin 4 receptor (MC4R) is a key player in hypothalamic weight regulation and energy expenditure as part of the leptin–melanocortin pathway. Mutations in this G protein coupled receptor (GPCR) are the most common cause for monogenetic obesity, which appears to be mediated by changes in the anorectic action of MC4R via GS-dependent cyclic adenosine-monophosphate (cAMP) signaling as well as other signaling pathways. To study potential bias in the effects of MC4R mutations between the different signaling pathways, we investigated three major MC4R mutations: a GS loss-of-function (S127L) and a GS gain-of-function mutant (H158R), as well as the most common European single nucleotide polymorphism (V103I). We tested signaling of all four major G protein families plus extracellular regulated kinase (ERK) phosphorylation and β-arrestin2 recruitment, using the two endogenous agonists, α- and β-melanocyte stimulating hormone (MSH), along with a synthetic peptide agonist (NDP-α-MSH). The S127L mutation led to a full loss-of-function in all investigated pathways, whereas V103I and H158R were clearly biased towards the Gq/11 pathway when challenged with the endogenous ligands. These results show that MC4R mutations can cause vastly different changes in the various MC4R signaling pathways and highlight the importance of a comprehensive characterization of receptor mutations.

scholarly journals The single pass membrane protein MRAP2 regulates energy homeostasis by promoting primary cilia localization of the G protein-coupled receptor MC4R

2020 ◽  
Author(s):  
Adélaïde Bernard ◽  
Irene Ojeda Naharros ◽  
Florence Bourgain-Guglielmetti ◽  
Jordi Ciprin ◽  
Xinyu Yue ◽  
...  
Keyword(s):  
G Protein ◽  
Energy Homeostasis ◽  
Primary Cilia ◽  
Melanocortin Receptor ◽  
G Protein Coupled Receptor ◽  
Promoting Effect ◽  
Receptor Associated Protein ◽  
Extracellular Signals ◽  
Melanocortin 4 Receptor ◽  
G Protein Coupled

ABSTRACTThe G protein-coupled receptor MC4R (Melanocortin-4 Receptor) and its associated protein MRAP2 (Melanocortin Receptor-Associated Protein 2) are both essential for the regulation of food intake and body weight in humans and mice. MC4R localizes and functions at the neuronal primary cilium, a microtubule-based organelle that senses and relays extracellular signals. Here, we demonstrate that MRAP2 is critical for the ciliary localization and weight-regulating function of MC4R. Our data reveal that GPCR localization to primary cilia can require specific accessory proteins that may not be present in heterologous cell systems. Our findings also demonstrate the essential role of neuronal primary cilia localization of MC4R for adequate control of energy homeostasis and the obesity-promoting effect of genetic disruption of this pathway.

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 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 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 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 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 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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