Identification of Protein Interactions by Yeast Two-Hybrid Screening and Coimmunoprecipitation

  • Michael Tanowitz
  • Mark von Zastrow
Part of the Methods in Molecular Biology book series (MIMB, volume 259)

Abstract

Many protein interactions with G-protein-coupled receptors (GPCRs) appear to influence receptor signaling and functional regulation. There is great interest therefore in methods for the identification of novel or unanticipated GPCR binding proteins. A proven method for identifying such protein interactions is the yeast two-hybrid screen, which involves screening the protein products of a cDNA library with a selected domain derived from a GPCR. Once it is established that a candidate protein produces a specific positive interaction within the yeast two-hybrid system, it is important to demonstrate further that this interaction is likely to occur in vivo. Coimmunoprecipitation, in which proteins of interest are copurified with the receptor under study, is a good way to address this important issue. Together, the yeast two-hybrid screen and coimmunoprecipitation are a useful way to identify and sort through candidate GPCR-interacting proteins prior to analysis in physiological studies.

Key Words

GAL4 system GPCR binding protein GPCR-protein interactions immunoprecipitation optimization interaction cloning receptor-protein crosslinking receptor-protein solubilization 

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Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  • Michael Tanowitz
    • 1
  • Mark von Zastrow
    • 1
  1. 1.Department of PsychiatryUniversity of CaliforniaSan Francisco

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