Utility of an "allosteric site-impaired" M2 muscarinic acetylcholine receptor as a novel construct for validating mechanisms of action of synthetic and putative endogenous allosteric modulators

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Abstract

Muscarinic acetylcholine receptors (mAChRs) are exemplar models for understanding G protein-coupled receptor (GPCR) allostery, possessing a "common" allosteric site in an extracellular vestibule (ECV) for synthetic modulators including gallamine, strychnine, and brucine. In addition, there is intriguing evidence of endogenous peptides/proteins that may target this region at the M2 mAChR. A common feature of synthetic and endogenous M2 mAChR negative allosteric modulators (NAMs) is their cationic nature. Using a structurebased approach, we previously designed a mutant M2 mAChR (N410K1T423K) to specifically abrogate binding of ECV cationic modulators (Dror et al., 2013). Herein, we used this "allosteric site-impaired" receptor to investigate allosteric interactions of synthetic modulators as well as basic peptides (poly-L-arginine, endogenously produced protamine, and major basic protein). Using [3H]N-methylscopolamine equilibrium and kinetic binding and functional assays of guanosine 5′-O- [γ-thio]triphosphate [35S] binding and extracellular signalregulated kinases 1 and 2 phosphorylation, we found modest effects of the mutations on potencies of orthosteric antagonists and an increase in the affinity of the cognate agonist, acetylcholine, likely reflecting the effect of the mutations on the access/egress of these ligands into the orthosteric pocket. More importantly, we noted a significant abrogation in affinity for all synthetic or peptidic modulators at the mutant mAChR, validating their allosteric nature. Collectively, these findings provide evidence for a hitherto-unappreciated role of endogenous cationic peptides interacting allosterically at the M2 mAChR and identify the allosteric site-impaired GPCR as a tool for validating NAM activity as well as a potential candidate for future chemogenetic strategies to understand the physiology of endogenous allosteric substances.

Original languageEnglish
Pages (from-to)1298-1309
Number of pages12
JournalMolecular Pharmacology
Volume94
Issue number5
DOIs
Publication statusPublished - 1 Nov 2018

Cite this

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title = "Utility of an {"}allosteric site-impaired{"} M2 muscarinic acetylcholine receptor as a novel construct for validating mechanisms of action of synthetic and putative endogenous allosteric modulators",
abstract = "Muscarinic acetylcholine receptors (mAChRs) are exemplar models for understanding G protein-coupled receptor (GPCR) allostery, possessing a {"}common{"} allosteric site in an extracellular vestibule (ECV) for synthetic modulators including gallamine, strychnine, and brucine. In addition, there is intriguing evidence of endogenous peptides/proteins that may target this region at the M2 mAChR. A common feature of synthetic and endogenous M2 mAChR negative allosteric modulators (NAMs) is their cationic nature. Using a structurebased approach, we previously designed a mutant M2 mAChR (N410K1T423K) to specifically abrogate binding of ECV cationic modulators (Dror et al., 2013). Herein, we used this {"}allosteric site-impaired{"} receptor to investigate allosteric interactions of synthetic modulators as well as basic peptides (poly-L-arginine, endogenously produced protamine, and major basic protein). Using [3H]N-methylscopolamine equilibrium and kinetic binding and functional assays of guanosine 5′-O- [γ-thio]triphosphate [35S] binding and extracellular signalregulated kinases 1 and 2 phosphorylation, we found modest effects of the mutations on potencies of orthosteric antagonists and an increase in the affinity of the cognate agonist, acetylcholine, likely reflecting the effect of the mutations on the access/egress of these ligands into the orthosteric pocket. More importantly, we noted a significant abrogation in affinity for all synthetic or peptidic modulators at the mutant mAChR, validating their allosteric nature. Collectively, these findings provide evidence for a hitherto-unappreciated role of endogenous cationic peptides interacting allosterically at the M2 mAChR and identify the allosteric site-impaired GPCR as a tool for validating NAM activity as well as a potential candidate for future chemogenetic strategies to understand the physiology of endogenous allosteric substances.",
author = "Moo, {Ee Von} and Sexton, {Patrick M.} and Arthur Christopoulos and Celine Valant",
year = "2018",
month = "11",
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doi = "10.1124/mol.118.112490",
language = "English",
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T1 - Utility of an "allosteric site-impaired" M2 muscarinic acetylcholine receptor as a novel construct for validating mechanisms of action of synthetic and putative endogenous allosteric modulators

AU - Moo, Ee Von

AU - Sexton, Patrick M.

AU - Christopoulos, Arthur

AU - Valant, Celine

PY - 2018/11/1

Y1 - 2018/11/1

N2 - Muscarinic acetylcholine receptors (mAChRs) are exemplar models for understanding G protein-coupled receptor (GPCR) allostery, possessing a "common" allosteric site in an extracellular vestibule (ECV) for synthetic modulators including gallamine, strychnine, and brucine. In addition, there is intriguing evidence of endogenous peptides/proteins that may target this region at the M2 mAChR. A common feature of synthetic and endogenous M2 mAChR negative allosteric modulators (NAMs) is their cationic nature. Using a structurebased approach, we previously designed a mutant M2 mAChR (N410K1T423K) to specifically abrogate binding of ECV cationic modulators (Dror et al., 2013). Herein, we used this "allosteric site-impaired" receptor to investigate allosteric interactions of synthetic modulators as well as basic peptides (poly-L-arginine, endogenously produced protamine, and major basic protein). Using [3H]N-methylscopolamine equilibrium and kinetic binding and functional assays of guanosine 5′-O- [γ-thio]triphosphate [35S] binding and extracellular signalregulated kinases 1 and 2 phosphorylation, we found modest effects of the mutations on potencies of orthosteric antagonists and an increase in the affinity of the cognate agonist, acetylcholine, likely reflecting the effect of the mutations on the access/egress of these ligands into the orthosteric pocket. More importantly, we noted a significant abrogation in affinity for all synthetic or peptidic modulators at the mutant mAChR, validating their allosteric nature. Collectively, these findings provide evidence for a hitherto-unappreciated role of endogenous cationic peptides interacting allosterically at the M2 mAChR and identify the allosteric site-impaired GPCR as a tool for validating NAM activity as well as a potential candidate for future chemogenetic strategies to understand the physiology of endogenous allosteric substances.

AB - Muscarinic acetylcholine receptors (mAChRs) are exemplar models for understanding G protein-coupled receptor (GPCR) allostery, possessing a "common" allosteric site in an extracellular vestibule (ECV) for synthetic modulators including gallamine, strychnine, and brucine. In addition, there is intriguing evidence of endogenous peptides/proteins that may target this region at the M2 mAChR. A common feature of synthetic and endogenous M2 mAChR negative allosteric modulators (NAMs) is their cationic nature. Using a structurebased approach, we previously designed a mutant M2 mAChR (N410K1T423K) to specifically abrogate binding of ECV cationic modulators (Dror et al., 2013). Herein, we used this "allosteric site-impaired" receptor to investigate allosteric interactions of synthetic modulators as well as basic peptides (poly-L-arginine, endogenously produced protamine, and major basic protein). Using [3H]N-methylscopolamine equilibrium and kinetic binding and functional assays of guanosine 5′-O- [γ-thio]triphosphate [35S] binding and extracellular signalregulated kinases 1 and 2 phosphorylation, we found modest effects of the mutations on potencies of orthosteric antagonists and an increase in the affinity of the cognate agonist, acetylcholine, likely reflecting the effect of the mutations on the access/egress of these ligands into the orthosteric pocket. More importantly, we noted a significant abrogation in affinity for all synthetic or peptidic modulators at the mutant mAChR, validating their allosteric nature. Collectively, these findings provide evidence for a hitherto-unappreciated role of endogenous cationic peptides interacting allosterically at the M2 mAChR and identify the allosteric site-impaired GPCR as a tool for validating NAM activity as well as a potential candidate for future chemogenetic strategies to understand the physiology of endogenous allosteric substances.

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U2 - 10.1124/mol.118.112490

DO - 10.1124/mol.118.112490

M3 - Article

VL - 94

SP - 1298

EP - 1309

JO - Molecular Pharmacology

JF - Molecular Pharmacology

SN - 1521-0111

IS - 5

ER -