Structural aspects of M₃ muscarinic acetylcholine receptor dimer formation and activation

FASEB J. 2012 Feb;26(2):604-16. doi: 10.1096/fj.11-191510. Epub 2011 Oct 26.


To explore the structural mechanisms underlying the assembly and activation of family A GPCR dimers, we used the rat M(3) muscarinic acetylcholine receptor (M3R) as a model system. Studies with Cys-substituted mutant M3Rs expressed in COS-7 cells led to the identification of several mutant M3Rs that exclusively existed as cross-linked dimers under oxidizing conditions. The cross-linked residues were located at the bottom of transmembrane domain 5 (TM5) and within the N-terminal portion of the third intracellular loop (i3 loop). Studies with urea-stripped membranes demonstrated that M3R disulfide cross-linking did not require the presence of heterotrimeric G proteins. Molecular modeling studies indicated that the cross-linking data were in excellent agreement with the existence of a low-energy M3R dimer characterized by a TM5-TM5 interface. [(35)S]GTPγS binding/Gα(q/11) immunoprecipitation assays revealed that an M3R dimer that was cross-linked within the N-terminal portion of the i3 loop (264C) was functionally severely impaired (∼50% reduction in receptor-G-protein coupling, as compared to control M3R). These data support the novel concept that agonist-induced activation of M3R dimers requires a conformational change of the N-terminal segment of the i3 loop. Given the high degree of structural homology among family A GPCRs, these findings should be of broad significance.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Animals
  • Binding Sites / genetics
  • COS Cells
  • Chlorocebus aethiops
  • Cross-Linking Reagents
  • Dimerization
  • GTP-Binding Protein alpha Subunits, Gq-G11 / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutant Proteins / chemistry
  • Mutant Proteins / genetics
  • Mutant Proteins / metabolism
  • Protein Structure, Quaternary
  • Rats
  • Receptor, Muscarinic M3 / chemistry*
  • Receptor, Muscarinic M3 / genetics
  • Receptor, Muscarinic M3 / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism


  • Cross-Linking Reagents
  • Mutant Proteins
  • Receptor, Muscarinic M3
  • Recombinant Proteins
  • GTP-Binding Protein alpha Subunits, Gq-G11