Muscarinic receptor family interacting proteins: role in receptor function

J Neurosci Methods. 2011 Feb 15;195(2):161-9. doi: 10.1016/j.jneumeth.2010.11.025. Epub 2010 Dec 4.


G protein-coupled receptors constitute one of the most important families of membrane receptors through which cells respond to extracellular stimuli. Receptors of this superfamily likely function as signal transduction complexes. The identification and analysis of their components provide new insights into a better understanding of these receptors' function and regulation. We used tandem-affinity purification and mass spectrometry as a systematic approach to characterize multiprotein complexes in the acetylcholine muscarinic receptor subfamily. To overcome the limitations associated with membrane protein receptor solubilization with detergents, we developed a strategy in which receptors are co-expressed with a cytoplasmic minigene construct, encoding the third intracellular loop and the C-terminal tail tagged to the tandem-affinity-cassette of each receptor subtype. Numerous protein complexes were identified, including many new interactions in various signalling pathways. Systematic identification data set together with protein interactions reported in the literature revealed a high degree of connectivity. These allow the proposal, for the first time, of an outline of the muscarinic interactome as a network of protein complexes and a context for a more reasoned and informed approach to drug discovery and muscarinic receptor subtype specificities.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • A Kinase Anchor Proteins / metabolism
  • Adaptor Proteins, Signal Transducing / metabolism
  • Cell Cycle Proteins / metabolism
  • Cell Line, Tumor
  • Chromatography, Affinity
  • Humans
  • Immunoprecipitation / methods
  • Mass Spectrometry / methods
  • Multiprotein Complexes / metabolism*
  • Neuroblastoma
  • Nonlinear Dynamics
  • Paxillin / metabolism
  • Protein Binding
  • Protein Phosphatase 2 / metabolism
  • Protein Structure, Tertiary
  • Receptors, Muscarinic / genetics
  • Receptors, Muscarinic / physiology*
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Transfection / methods


  • A Kinase Anchor Proteins
  • Adaptor Proteins, Signal Transducing
  • Cell Cycle Proteins
  • GIT1 protein, human
  • Multiprotein Complexes
  • Paxillin
  • Receptors, Muscarinic
  • Protein Phosphatase 2