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Tandem Affinity Purification and Identification of Heterotrimeric G Protein-Associated Proteins

  • Syed M. Ahmed
  • Avais M. Daulat
  • Stéphane AngersEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 756)

Abstract

Heterotrimeric G proteins are the main signal-transducing molecules activated by G protein-coupled receptors. Their GTP-dependent activation leads to the regulation of different effectors such as adenylyl cyclases, phospholipases, and RhoGEFs. To understand the full biological consequences of GPCR signalling and to further understand the cross-talk with other signalling pathways, the complement of proteins associating with heterotrimeric G proteins needs to be identified. Here we describe our mass spectrometry-based proteomic approaches for the study of Gβγ and Gα protein complexes. This approach is predicated on the establishment of mammalian cell lines constitutively or inducibly expressing affinity-tagged versions of Gβγ or wild-type and constitutively active Gα subunits, respectively.

Key words

Heterotrimeric G protein Tandem affinity purification Proteomics Protein complex Gα subunit Gβγ subunit 

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Syed M. Ahmed
    • 1
  • Avais M. Daulat
    • 2
  • Stéphane Angers
    • 2
    Email author
  1. 1.Department of Pharmaceutical Sciences & Biochemistry, Leslie Dan Faculty of PharmacyUniversity of TorontoTorontoCanada
  2. 2.Department of Pharmaceutical Sciences, Leslie Dan Faculty of PharmacyUniversity of TorontoTorontoCanada

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