scholarly journals Uveal Melanoma Oncogene CYSLTR2 Encodes a Constitutively Active GPCR Highly Biased Toward Gq Signaling

2019 ◽  
Author(s):  
Emilie Ceraudo ◽  
Mizuho Horioka ◽  
Jordan M. Mattheisen ◽  
Tyler D. Hitchman ◽  
Amanda R. Moore ◽  
...  
Keyword(s):  
G Protein ◽  
Uveal Melanoma ◽  
Transmembrane Helix ◽  
Single Amino Acid ◽  
Constitutive Activity ◽  
Mutant Receptor ◽  
Biased Signaling ◽  
Cysteinyl Leukotriene Receptor ◽  
Beta Arrestins ◽  
Constitutively Active

AbstractThe G protein-coupled receptor (GPCR) cysteinyl-leukotriene receptor 2 (CysLTR2) with a single amino acid mutation at position 3.43 (Leu replaced with Gln at position 129 in transmembrane helix 3) causes uveal melanoma in humans. The ability of CysLTR2-L129Q to cause malignant transformation has been hypothesized to result from constitutive activity. We show that CysLTR2-L129Q is a constitutively active mutant (CAM) that strongly drives Gq/11 signaling pathways in melan-a melanocytes and in HEK293T cells in culture. However, the mutant receptor only very weakly recruits beta-arrestins 1 and 2. The mutant receptor displays profound signaling bias while avoiding arrestin-mediated downregulation. The mechanism of the signaling bias results from the creation of a hydrogen-bond network that stabilizes the active G protein signaling state through novel interactions with the highly-conserved NPxxY motif on helix 7. Furthermore, the mutation destabilizes a putative allosteric sodium-binding site that usually stabilizes the inactive state of GPCRs. Thus, the mutation has a dual role of promoting the active state while destabilizing inactivating allosteric networks. The high degree of constitutive activity renders existing orthosteric antagonist ligands of CysLTR2 ineffective as inverse agonists of the mutant. CysLTR2 is the first example of a GPCR oncogene that encodes a GPCR with constitutive highly biased signaling that can escape cellular downregulation mechanisms.

scholarly journals Direct evidence that the GPCR CysLTR2 mutant causative of uveal melanoma is constitutively active with highly biased signaling

2020 ◽  
pp. jbc.RA120.015352
Author(s):  
Emilie Ceraudo ◽  
Mizuho Horioka ◽  
Jordan M Mattheisen ◽  
Tyler D Hitchman ◽  
Amanda R. Moore ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Uveal Melanoma ◽  
Constitutive Activity ◽  
Gene Encoding ◽  
Cysteinyl Leukotriene ◽  
Activating Mutation ◽  
Biased Signaling ◽  
Oncogenic Driver ◽  
Cysteinyl Leukotriene Receptor ◽  
Constitutively Active

Uveal melanoma is the most common eye cancer in adults and is clinically and genetically distinct from skin cutaneous melanoma. In a subset of cases, the oncogenic driver is an activating mutation in CYSLTR2, the gene encoding the G protein-coupled receptor (GPCR) cysteinyl-leukotriene receptor 2. The mutant CYSLTR2 encodes for CysLTR2-L129Q receptor, with the substitution of Leu to Gln at position 129 (3.43). The ability of CysLTR2-L129Q to cause malignant transformation has been hypothesized to result from constitutive activity, but how the receptor could escape desensitization is unknown. Here we characterize the functional properties of CysLTR2-L129Q. We show that CysLTR2-L129Q is a constitutively active mutant that strongly drives Gq/11 signaling pathways. However, CysLTR2-L129Q only poorly recruits β-arrestin. Using a modified Slack-Hall operational model, we quantified the constitutive activity for both pathways and conclude that CysLTR2-L129Q displays profound signaling bias for Gq/11 signaling pathways while escaping β-arrestin-mediated downregulation. CYSLTR2 is the first known example of a GPCR driver oncogene that encodes a highly biased constitutively active mutant receptor. These results provide new insights into the mechanism of CysLTR2-L129Q oncoprotein signaling and suggest CYSLTR2 as a promising potential therapeutic target in uveal melanoma.

Identification of a Potent Inverse Agonist at a Constitutively Active Mutant of Human P2Y12 Receptor.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3942-3942
Author(s):  
Zhongren Ding ◽  
Soochong Kim ◽  
Satya P. Kunapuli
Keyword(s):  
G Protein ◽  
Pertussis Toxin ◽  
Human Platelet ◽  
P2y Receptors ◽  
Inverse Agonist ◽  
Human Platelets ◽  
P2y12 Receptor ◽  
Constitutive Activity ◽  
P2y12 Antagonists ◽  
Constitutively Active

Abstract Human platelets express two P2Y receptors: Gq-coupled P2Y1 and Gi-coupled P2Y12. Both P2Y1 and P2Y12 are ADP receptors on human platelets and are essential for ADP-induced platelet aggregation that plays pivotal roles in thrombosis and hemostasis. Numerous constitutively active G protein-coupled receptors have been described in natural or recombinant systems but in the P2Y receptors, to date, no constitutive activity has been reported. In our effort to identify G protein coupling domains of human platelet ADP receptor we constructed a chimeric HA-tagged human P2Y12 receptor with its C-terminus replaced by the corresponding part of human P2Y1 receptor and stably expressed it in CHO-K1 cells. Interestingly, the chimeric P2Y12 mutant exhibited a high level of constitutive activity as evidenced by decreased cAMP levels in the absence of agonists. The constitutive activation of the chimeric P2Y12 mutant was abolished by pertussis toxin, a Gi inhibitor. The constitutively active P2Y12 mutant retained normal responses to 2-MeSADP, with an EC50 of 0.15 ± 0.04 nM. The constitutively active P2Y12 mutant caused Akt phosphorylation that was abolished by the addition of pertussis toxin. Pharmacological evaluation of several P2Y12 antagonists revealed AR-C78511 as a potent P2Y12 inverse agonist whereas AR-C69931MX as a neutral antagonist. In conclusion, this is the first report of a cell line stably expressing a constitutively active mutant of human platelet P2Y12 receptor and the identification of potent inverse agonist.

scholarly journals Structures of active melanocortin-4 receptor–Gs-protein complexes with NDP-α-MSH and setmelanotide

Cell Research ◽  
2021 ◽  
Author(s):  
Nicolas A. Heyder ◽  
Gunnar Kleinau ◽  
David Speck ◽  
Andrea Schmidt ◽  
Sarah Paisdzior ◽  
...  
Keyword(s):  
G Protein ◽  
Energy Homeostasis ◽  
Protein Complexes ◽  
Transmembrane Helix ◽  
Receptor Activation ◽  
Binding Modes ◽  
Intracellular Loop ◽  
Melanocortin 4 Receptor ◽  
Biased Signaling ◽  
Gs Protein

AbstractThe melanocortin-4 receptor (MC4R), a hypothalamic master regulator of energy homeostasis and appetite, is a class A G-protein-coupled receptor and a prime target for the pharmacological treatment of obesity. Here, we present cryo-electron microscopy structures of MC4R–Gs-protein complexes with two drugs recently approved by the FDA, the peptide agonists NDP-α-MSH and setmelanotide, with 2.9 Å and 2.6 Å resolution. Together with signaling data from structure-derived MC4R mutants, the complex structures reveal the agonist-induced origin of transmembrane helix (TM) 6-regulated receptor activation. The ligand-binding modes of NDP-α-MSH, a high-affinity linear variant of the endogenous agonist α-MSH, and setmelanotide, a cyclic anti-obesity drug with biased signaling toward Gq/11, underline the key role of TM3 in ligand-specific interactions and of calcium ion as a ligand-adaptable cofactor. The agonist-specific TM3 interplay subsequently impacts receptor–Gs-protein interfaces at intracellular loop 2, which also regulates the G-protein coupling profile of this promiscuous receptor. Finally, our structures reveal mechanistic details of MC4R activation/inhibition, and provide important insights into the regulation of the receptor signaling profile which will facilitate the development of tailored anti-obesity drugs.

scholarly journals Structural dynamics and energetics underlying allosteric inactivation of the cannabinoid receptor CB1

2015 ◽  
Vol 112 (27) ◽  
pp. 8469-8474 ◽  
Author(s):  
Jonathan F. Fay ◽  
David L. Farrens
Keyword(s):  
G Protein ◽  
Cannabinoid Receptor ◽  
Transmembrane Helix ◽  
Binding Pocket ◽  
Fluorescence Labeling ◽  
Allosteric Site ◽  
Binding Studies ◽  
Protein Activation ◽  
G Protein Activation ◽  
Biased Signaling

G protein-coupled receptors (GPCRs) are surprisingly flexible molecules that can do much more than simply turn on G proteins. Some even exhibit biased signaling, wherein the same receptor preferentially activates different G-protein or arrestin signaling pathways depending on the type of ligand bound. Why this behavior occurs is still unclear, but it can happen with both traditional ligands and ligands that bind allosterically outside the orthosteric receptor binding pocket. Here, we looked for structural mechanisms underlying these phenomena in the marijuana receptor CB1. Our work focused on the allosteric ligand Org 27569, which has an unusual effect on CB1—it simultaneously increases agonist binding, decreases G-protein activation, and induces biased signaling. Using classical pharmacological binding studies, we find that Org 27569 binds to a unique allosteric site on CB1 and show that it can act alone (without need for agonist cobinding). Through mutagenesis studies, we find that the ability of Org 27569 to bind is related to how much receptor is in an active conformation that can couple with G protein. Using these data, we estimated the energy differences between the inactive and active states. Finally, site-directed fluorescence labeling studies show the CB1 structure stabilized by Org 27569 is different and unique from that stabilized by antagonist or agonist. Specifically, transmembrane helix 6 (TM6) movements associated with G-protein activation are blocked, but at the same time, helix 8/TM7 movements are enhanced, suggesting a possible mechanism for the ability of Org 27569 to induce biased signaling.

scholarly journals Identification of a Polar Region in Transmembrane Domain 6 That Regulates the Function of the G Protein-Coupled α-Factor Receptor

1998 ◽  
Vol 18 (12) ◽  
pp. 7205-7215 ◽  
Author(s):  
Peter Dube ◽  
James B. Konopka
Keyword(s):  
G Protein ◽  
Transmembrane Domain ◽  
Transmembrane Helix ◽  
Fold Increase ◽  
Intramolecular Interactions ◽  
Constitutive Activity ◽  
Potential Contact ◽  
Contact Residues ◽  
Hydrophobic Amino Acid Residue ◽  
G Protein Coupled

ABSTRACT The α-factor pheromone receptor (Ste2p) of the yeastSaccharomyces cerevisiae belongs to the family of G protein-coupled receptors that contain seven transmembrane domains (TMDs). Because polar residues can influence receptor structure by forming intramolecular contacts between TMDs, we tested the role of the five polar amino acids in TMD6 of the α-factor receptor by mutating these residues to nonpolar leucine. Interestingly, a subset of these mutants showed increased affinity for ligand and constitutive receptor activity. The mutation of the most polar residue, Q253L, resulted in 25-fold increased affinity and a 5-fold-higher basal level of signaling that was equal to about 19% of the α-factor induced maximum signal. Mutation of the adjacent residue, S254L, caused weaker constitutive activity and a 5-fold increase in affinity. Comparison of nine different mutations affecting Ser254 showed that an S254F mutation caused higher constitutive activity, suggesting that a large hydrophobic amino acid residue at position 254 alters transmembrane helix packing. Thus, these studies indicate that Gln253 and Ser254 are likely to be involved in intramolecular interactions with other TMDs. Furthermore, Gln253 and Ser254 fall on one side of the transmembrane helix that is on the opposite side from residues that do not cause constitutive activity when mutated. These results suggest that Gln253and Ser254 face inward toward the other TMDs and thus provide the first experimental evidence to suggest the orientation of a TMD in this receptor. Consistent with this, we identified two residues in TMD7 (Ser288 and Ser292) that are potential contact residues for Gln253 because mutations affecting these residues also cause constitutive activity. Altogether, these results identify a new domain of the α-factor receptor that regulates its ability to enter the activated conformation.

GPCR structure and function relationship: identification of a biased apelin receptor mutant

2018 ◽  
Vol 475 (23) ◽  
pp. 3813-3826 ◽  
Author(s):  
Ting Ban ◽  
Xun Li ◽  
Xiaochuan Ma ◽  
Hui Yang ◽  
Yunpeng Song ◽  
...  
Keyword(s):  
Ligand Binding ◽  
G Protein ◽  
Molecular Mechanism ◽  
Transmembrane Domain ◽  
Hydrophobic Interactions ◽  
Double Mutation ◽  
Mutant Receptor ◽  
Therapeutic Benefits ◽  
Biased Signaling ◽  
Apelin Receptor

Biased ligands of G protein-coupled receptors (GPCRs) may have improved therapeutic benefits and safety profiles. However, the molecular mechanism of GPCR biased signaling remains largely unknown. Using apelin receptor (APJ) as a model, we systematically investigated the potential effects of amino acid residues around the orthosteric binding site on biased signaling. We discovered that a single residue mutation I109A (I1093.32) in the transmembrane domain 3 (TM3) located in the deep ligand-binding pocket was sufficient to convert a balanced APJ into a G protein signaling biased receptor. APJ I109A mutant receptor retained full capabilities in ligand binding and G protein activation, but was defective in GRK recruitment, β-arrestin recruitment, and downstream receptor-mediated ERK activation. Based on molecular dynamics simulations, we proposed a molecular mechanism for biased signaling of I109A mutant receptor. We postulate that due to the extra space created by I109A mutation, the phenyl group of the last residue (Phe-13) of apelin rotates down and initiates a cascade of conformational changes in TM3. Phe-13 formed a new cluster of hydrophobic interactions with the sidechains of residues in TM3, including F1103.33 and M1133.36, which stabilizes the mutant receptor in a conformation favoring biased signaling. Interruption of these stabilizing interactions by double mutation F110A/I109A or M113A/I109A largely restored the β-arrestin-mediated signaling. Taken together, we describe herein the discovery of a biased APJ mutant receptor and provide detailed molecular insights into APJ signaling selectivity, facilitating the discovery of novel therapeutics targeting APJ.

Involvement of the constitutive activity of the GHS-R1a (ghrelin G-protein coupled receptor) in the tumorigenesis of somatotroph adenomas

2013 ◽  
Author(s):  
Yves Louis Mear ◽  
Xavier Come Donato ◽  
Marie Pierre Blanchard ◽  
Celine Defilles ◽  
Christophe Lisbonis ◽  
...  
Keyword(s):  
G Protein ◽  
Constitutive Activity ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

scholarly journals Constitutive Activity among Orphan Class-A G Protein Coupled Receptors

PLoS ONE ◽  
2015 ◽  
Vol 10 (9) ◽  
pp. e0138463 ◽  
Author(s):  
Adam L. Martin ◽  
Michael A. Steurer ◽  
Robert S. Aronstam
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Constitutive Activity ◽  
Class A ◽  
G Protein Coupled
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