Journal
JOURNAL OF NEUROSCIENCE METHODS
Volume 195, Issue 2, Pages 161-169Publisher
ELSEVIER
DOI: 10.1016/j.jneumeth.2010.11.025
Keywords
Muscarinic receptors; Interacting proteins; Tandem affinity purification; Signal network; G protein coupled receptors
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Funding
- MICINN [SAF2005-0848-C04-02, SAF2008-04943-C02-02]
- Ministerio de Ciencia e Innovacion [SAF2008-01462, CSD2R008-00005]
- European Social Foundation [FI2004]
- Catalonian Government [BE-2006]
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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. (C) 2010 Elsevier B.V. All rights reserved.
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