Identification of Protein Interactions by Yeast Two-Hybrid Screening and Coimmunoprecipitation
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 WordsGAL4 system GPCR binding protein GPCR-protein interactions immunoprecipitation optimization interaction cloning receptor-protein crosslinking receptor-protein solubilization
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