Identification of monellin as the first naturally derived proteinaceous allosteric agonist of metabotropic glutamate receptor 5

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Abstract

Allosteric modulators bind sites distinct from orthosteric ligands, allowing for improved spatiotemporal control of receptors and greater subtype selectivity. However, we recently showed that allosteric ligands previously classified as selective for select Class C G protein-coupled receptors (GPCRs) had unappreciated activity at other off-target receptors, in some cases higher affinity, within the class. Here, we extended our investigation of off-target activity of “selective” allosteric ligands for the sweet taste receptor. Using metabotropic glutamate receptor 5 (mGlu 5 ) as a representative of Class C GPCR, we assessed the sweet protein, monellin and the small-molecule artificial sweetener, NHDC. We found that monellin, but not NHDC, is an agonist for mGlu 5 . Radioligand binding and functional assays performed in cells expressing N-terminally truncated mGlu 5 demonstrated that monellin agonism was not mediated via the “common” allosteric binding site in the transmembrane domain but required the presence of the large extracellular N-terminal domain of mGlu 5 . Monellin displayed neutral functional cooperativity with orthosteric ligands. However, monellin positively modulated the mGlu 5 PAM-agonist, VU0424465, activity in intracellular calcium assays, but the interaction was neutral in inositol phosphate accumulation assays. Furthermore, monellin mGlu 5 agonism was positively modulated by the mGlu 5 pure PAM, VU0360172. Taken together, these data indicate that monellin is an allosteric agonist for mGlu 5 , binding to an allosteric binding site on the N-terminus that is functionally linked to the common Class C GPCR allosteric site in a biased manner. This is the first evidence of a naturally derived proteinaceous allosteric ligand for the mGlu receptor family.

Original languageEnglish
Number of pages22
JournalBasic and Clinical Pharmacology and Toxicology
DOIs
Publication statusAccepted/In press - 14 Apr 2019

Keywords

  • allosteric modulator
  • biased modulation
  • G protein-coupled receptor
  • sweet protein

Cite this

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title = "Identification of monellin as the first naturally derived proteinaceous allosteric agonist of metabotropic glutamate receptor 5",
abstract = "Allosteric modulators bind sites distinct from orthosteric ligands, allowing for improved spatiotemporal control of receptors and greater subtype selectivity. However, we recently showed that allosteric ligands previously classified as selective for select Class C G protein-coupled receptors (GPCRs) had unappreciated activity at other off-target receptors, in some cases higher affinity, within the class. Here, we extended our investigation of off-target activity of “selective” allosteric ligands for the sweet taste receptor. Using metabotropic glutamate receptor 5 (mGlu 5 ) as a representative of Class C GPCR, we assessed the sweet protein, monellin and the small-molecule artificial sweetener, NHDC. We found that monellin, but not NHDC, is an agonist for mGlu 5 . Radioligand binding and functional assays performed in cells expressing N-terminally truncated mGlu 5 demonstrated that monellin agonism was not mediated via the “common” allosteric binding site in the transmembrane domain but required the presence of the large extracellular N-terminal domain of mGlu 5 . Monellin displayed neutral functional cooperativity with orthosteric ligands. However, monellin positively modulated the mGlu 5 PAM-agonist, VU0424465, activity in intracellular calcium assays, but the interaction was neutral in inositol phosphate accumulation assays. Furthermore, monellin mGlu 5 agonism was positively modulated by the mGlu 5 pure PAM, VU0360172. Taken together, these data indicate that monellin is an allosteric agonist for mGlu 5 , binding to an allosteric binding site on the N-terminus that is functionally linked to the common Class C GPCR allosteric site in a biased manner. This is the first evidence of a naturally derived proteinaceous allosteric ligand for the mGlu receptor family.",
keywords = "allosteric modulator, biased modulation, G protein-coupled receptor, sweet protein",
author = "Chen, {Amy N.Y.} and Hellyer, {Shane D.} and Trinh, {Phuc N.H.} and Katie Leach and Gregory, {Karen J.}",
year = "2019",
month = "4",
day = "14",
doi = "10.1111/bcpt.13239",
language = "English",
journal = "Basic and Clinical Pharmacology and Toxicology",
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TY - JOUR

T1 - Identification of monellin as the first naturally derived proteinaceous allosteric agonist of metabotropic glutamate receptor 5

AU - Chen, Amy N.Y.

AU - Hellyer, Shane D.

AU - Trinh, Phuc N.H.

AU - Leach, Katie

AU - Gregory, Karen J.

PY - 2019/4/14

Y1 - 2019/4/14

N2 - Allosteric modulators bind sites distinct from orthosteric ligands, allowing for improved spatiotemporal control of receptors and greater subtype selectivity. However, we recently showed that allosteric ligands previously classified as selective for select Class C G protein-coupled receptors (GPCRs) had unappreciated activity at other off-target receptors, in some cases higher affinity, within the class. Here, we extended our investigation of off-target activity of “selective” allosteric ligands for the sweet taste receptor. Using metabotropic glutamate receptor 5 (mGlu 5 ) as a representative of Class C GPCR, we assessed the sweet protein, monellin and the small-molecule artificial sweetener, NHDC. We found that monellin, but not NHDC, is an agonist for mGlu 5 . Radioligand binding and functional assays performed in cells expressing N-terminally truncated mGlu 5 demonstrated that monellin agonism was not mediated via the “common” allosteric binding site in the transmembrane domain but required the presence of the large extracellular N-terminal domain of mGlu 5 . Monellin displayed neutral functional cooperativity with orthosteric ligands. However, monellin positively modulated the mGlu 5 PAM-agonist, VU0424465, activity in intracellular calcium assays, but the interaction was neutral in inositol phosphate accumulation assays. Furthermore, monellin mGlu 5 agonism was positively modulated by the mGlu 5 pure PAM, VU0360172. Taken together, these data indicate that monellin is an allosteric agonist for mGlu 5 , binding to an allosteric binding site on the N-terminus that is functionally linked to the common Class C GPCR allosteric site in a biased manner. This is the first evidence of a naturally derived proteinaceous allosteric ligand for the mGlu receptor family.

AB - Allosteric modulators bind sites distinct from orthosteric ligands, allowing for improved spatiotemporal control of receptors and greater subtype selectivity. However, we recently showed that allosteric ligands previously classified as selective for select Class C G protein-coupled receptors (GPCRs) had unappreciated activity at other off-target receptors, in some cases higher affinity, within the class. Here, we extended our investigation of off-target activity of “selective” allosteric ligands for the sweet taste receptor. Using metabotropic glutamate receptor 5 (mGlu 5 ) as a representative of Class C GPCR, we assessed the sweet protein, monellin and the small-molecule artificial sweetener, NHDC. We found that monellin, but not NHDC, is an agonist for mGlu 5 . Radioligand binding and functional assays performed in cells expressing N-terminally truncated mGlu 5 demonstrated that monellin agonism was not mediated via the “common” allosteric binding site in the transmembrane domain but required the presence of the large extracellular N-terminal domain of mGlu 5 . Monellin displayed neutral functional cooperativity with orthosteric ligands. However, monellin positively modulated the mGlu 5 PAM-agonist, VU0424465, activity in intracellular calcium assays, but the interaction was neutral in inositol phosphate accumulation assays. Furthermore, monellin mGlu 5 agonism was positively modulated by the mGlu 5 pure PAM, VU0360172. Taken together, these data indicate that monellin is an allosteric agonist for mGlu 5 , binding to an allosteric binding site on the N-terminus that is functionally linked to the common Class C GPCR allosteric site in a biased manner. This is the first evidence of a naturally derived proteinaceous allosteric ligand for the mGlu receptor family.

KW - allosteric modulator

KW - biased modulation

KW - G protein-coupled receptor

KW - sweet protein

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U2 - 10.1111/bcpt.13239

DO - 10.1111/bcpt.13239

M3 - Article

JO - Basic and Clinical Pharmacology and Toxicology

JF - Basic and Clinical Pharmacology and Toxicology

SN - 1742-7835

ER -