Palmitoylation of muscarinic acetylcholine receptor m2 subtypes

Reduction in their ability to activate G proteins by mutation of a putative palmitoylation site, cysteine 457, in the carboxyl-terminal tail

Mariko Kato Hayashi, Tatsuya Haga

Research output: Contribution to journalArticle

59 Citations (Scopus)

Abstract

A putative palmitoylation site, Cys457, of muscarinic acetylcholine receptor m2 subtype (m2 receptor) was eliminated by conversion to alanine or stop codon by site-directed mutagenesis. The mutant m2 receptor C457A was not metabolically labeled with [3H]palmitic acid when expressed in Sf9 cells, whereas the wild-type m2 receptor was labeled under the same conditions. These results confirm that the Cys457 is the palmitoylation site. The rate of palmitoylation was markedly accelerated by addition of agonist, indicating that the palmitoylation reaction is affected by conformational changes of the receptor induced by agonist binding. The m2 receptor mutants without palmitoylation were purified and reconstituted with G proteins into phospholipid vesicles. Both mutants were good substrates of G protein- coupled receptor kinase 2 and the phosphorylation was stimulated by agonist and G protein βγ subunits, as was the case for wild-type receptors. The mutant receptors interacted with and activate G(i2) and G(o). However, the rate of [35]GTPγS binding to G(i2) was half as much for the mutants as that for the wild type, and the proportion of guanine nucleotide-sensitive high-affinity agonist binding sites was significantly less for mutants (42- 42%) compared to wild type (62%). These results indicate that the palmitoylation of m2 receptors is not an absolute requirement for their interaction with G proteins but enhances the ability of the receptors to interact with G proteins.

Original languageEnglish
Pages (from-to)376-382
Number of pages7
JournalArchives of Biochemistry and Biophysics
Volume340
Issue number2
DOIs
Publication statusPublished - 1997 Apr 15
Externally publishedYes

Fingerprint

Muscarinic M2 Receptors
Lipoylation
Muscarinic Receptors
GTP-Binding Proteins
Cysteine
Mutation
G-Protein-Coupled Receptor Kinase 2
Mutagenesis
Phosphorylation
Guanine Nucleotides
Palmitic Acid
Protein Subunits
Alanine
Sf9 Cells
Phospholipids
Terminator Codon
Binding Sites
Site-Directed Mutagenesis
Substrates

Keywords

  • acetylcholine
  • G protein
  • muscarinic
  • palmitoylation
  • phosphorylation

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

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title = "Palmitoylation of muscarinic acetylcholine receptor m2 subtypes: Reduction in their ability to activate G proteins by mutation of a putative palmitoylation site, cysteine 457, in the carboxyl-terminal tail",
abstract = "A putative palmitoylation site, Cys457, of muscarinic acetylcholine receptor m2 subtype (m2 receptor) was eliminated by conversion to alanine or stop codon by site-directed mutagenesis. The mutant m2 receptor C457A was not metabolically labeled with [3H]palmitic acid when expressed in Sf9 cells, whereas the wild-type m2 receptor was labeled under the same conditions. These results confirm that the Cys457 is the palmitoylation site. The rate of palmitoylation was markedly accelerated by addition of agonist, indicating that the palmitoylation reaction is affected by conformational changes of the receptor induced by agonist binding. The m2 receptor mutants without palmitoylation were purified and reconstituted with G proteins into phospholipid vesicles. Both mutants were good substrates of G protein- coupled receptor kinase 2 and the phosphorylation was stimulated by agonist and G protein βγ subunits, as was the case for wild-type receptors. The mutant receptors interacted with and activate G(i2) and G(o). However, the rate of [35]GTPγS binding to G(i2) was half as much for the mutants as that for the wild type, and the proportion of guanine nucleotide-sensitive high-affinity agonist binding sites was significantly less for mutants (42- 42{\%}) compared to wild type (62{\%}). These results indicate that the palmitoylation of m2 receptors is not an absolute requirement for their interaction with G proteins but enhances the ability of the receptors to interact with G proteins.",
keywords = "acetylcholine, G protein, muscarinic, palmitoylation, phosphorylation",
author = "Hayashi, {Mariko Kato} and Tatsuya Haga",
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AU - Haga, Tatsuya

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N2 - A putative palmitoylation site, Cys457, of muscarinic acetylcholine receptor m2 subtype (m2 receptor) was eliminated by conversion to alanine or stop codon by site-directed mutagenesis. The mutant m2 receptor C457A was not metabolically labeled with [3H]palmitic acid when expressed in Sf9 cells, whereas the wild-type m2 receptor was labeled under the same conditions. These results confirm that the Cys457 is the palmitoylation site. The rate of palmitoylation was markedly accelerated by addition of agonist, indicating that the palmitoylation reaction is affected by conformational changes of the receptor induced by agonist binding. The m2 receptor mutants without palmitoylation were purified and reconstituted with G proteins into phospholipid vesicles. Both mutants were good substrates of G protein- coupled receptor kinase 2 and the phosphorylation was stimulated by agonist and G protein βγ subunits, as was the case for wild-type receptors. The mutant receptors interacted with and activate G(i2) and G(o). However, the rate of [35]GTPγS binding to G(i2) was half as much for the mutants as that for the wild type, and the proportion of guanine nucleotide-sensitive high-affinity agonist binding sites was significantly less for mutants (42- 42%) compared to wild type (62%). These results indicate that the palmitoylation of m2 receptors is not an absolute requirement for their interaction with G proteins but enhances the ability of the receptors to interact with G proteins.

AB - A putative palmitoylation site, Cys457, of muscarinic acetylcholine receptor m2 subtype (m2 receptor) was eliminated by conversion to alanine or stop codon by site-directed mutagenesis. The mutant m2 receptor C457A was not metabolically labeled with [3H]palmitic acid when expressed in Sf9 cells, whereas the wild-type m2 receptor was labeled under the same conditions. These results confirm that the Cys457 is the palmitoylation site. The rate of palmitoylation was markedly accelerated by addition of agonist, indicating that the palmitoylation reaction is affected by conformational changes of the receptor induced by agonist binding. The m2 receptor mutants without palmitoylation were purified and reconstituted with G proteins into phospholipid vesicles. Both mutants were good substrates of G protein- coupled receptor kinase 2 and the phosphorylation was stimulated by agonist and G protein βγ subunits, as was the case for wild-type receptors. The mutant receptors interacted with and activate G(i2) and G(o). However, the rate of [35]GTPγS binding to G(i2) was half as much for the mutants as that for the wild type, and the proportion of guanine nucleotide-sensitive high-affinity agonist binding sites was significantly less for mutants (42- 42%) compared to wild type (62%). These results indicate that the palmitoylation of m2 receptors is not an absolute requirement for their interaction with G proteins but enhances the ability of the receptors to interact with G proteins.

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