Homologous mutations near the junction of the sixth transmembrane domain and the third extracellular loop lead to constitutive activity and enhanced agonist affinity at all muscarinic receptor subtypes

Diane J. Ford, Anthony Essex, Tracy A. Spalding, Ethan S. Burstein, John Ellis

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Abstract

Previous studies have found that a mutation near the junction of the sixth transmembrane domain (TM6) and the third extracellular loop of the M5 muscarinic receptor leads to constitutive activation and enhanced agonist affinity for the mutated receptor. These results were consistent with the extended ternary complex model, which predicts a correlation between agonist affinity and constitutive activity. We have introduced the homologous mutation into all five subtypes of the highly conserved muscarinic receptor family; SerThr→TyrPro was introduced into M1 and M5 and AsnThr→TyrPro was introduced into M2, M3, and M4. In binding assays, these mutations produced increases in affinities toward acetylcholine and carbachol that ranged from 5-fold at the M2 receptor to 15- to 20-fold at M1, M3, and M4, to 40-fold at M5. In functional assays, all five mutant receptors exhibited constitutive activity, at levels ranging between 30 and 80% of the maximal response elicited by carbachol. In every case, the muscarinic antagonist atropine inhibited this constitutive activity with high affinity. Thus, despite differences in effector coupling and in wild-type sequence at the mutation site, all five subtypes were activated by this mutation at the top of TM6. Previous studies of the M5 subtype have indicated that TM6 is a ligand-dependent switch that sets the activation state of the receptor. Based on the results of the present study, it is possible that TM6 represents a general switch for the activation of the muscarinic receptor family.

Original languageEnglish (US)
Pages (from-to)810-817
Number of pages8
JournalJournal of Pharmacology and Experimental Therapeutics
Volume300
Issue number3
DOIs
StatePublished - Mar 4 2002

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Muscarinic Receptors
Mutation
Carbachol
Muscarinic M5 Receptors
Muscarinic Antagonists
Atropine
Acetylcholine
Ligands

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Pharmacology

Cite this

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title = "Homologous mutations near the junction of the sixth transmembrane domain and the third extracellular loop lead to constitutive activity and enhanced agonist affinity at all muscarinic receptor subtypes",
abstract = "Previous studies have found that a mutation near the junction of the sixth transmembrane domain (TM6) and the third extracellular loop of the M5 muscarinic receptor leads to constitutive activation and enhanced agonist affinity for the mutated receptor. These results were consistent with the extended ternary complex model, which predicts a correlation between agonist affinity and constitutive activity. We have introduced the homologous mutation into all five subtypes of the highly conserved muscarinic receptor family; SerThr→TyrPro was introduced into M1 and M5 and AsnThr→TyrPro was introduced into M2, M3, and M4. In binding assays, these mutations produced increases in affinities toward acetylcholine and carbachol that ranged from 5-fold at the M2 receptor to 15- to 20-fold at M1, M3, and M4, to 40-fold at M5. In functional assays, all five mutant receptors exhibited constitutive activity, at levels ranging between 30 and 80{\%} of the maximal response elicited by carbachol. In every case, the muscarinic antagonist atropine inhibited this constitutive activity with high affinity. Thus, despite differences in effector coupling and in wild-type sequence at the mutation site, all five subtypes were activated by this mutation at the top of TM6. Previous studies of the M5 subtype have indicated that TM6 is a ligand-dependent switch that sets the activation state of the receptor. Based on the results of the present study, it is possible that TM6 represents a general switch for the activation of the muscarinic receptor family.",
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T1 - Homologous mutations near the junction of the sixth transmembrane domain and the third extracellular loop lead to constitutive activity and enhanced agonist affinity at all muscarinic receptor subtypes

AU - Ford, Diane J.

AU - Essex, Anthony

AU - Spalding, Tracy A.

AU - Burstein, Ethan S.

AU - Ellis, John

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