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  • Title: Visualization and quantification of GPCR trafficking in mammalian cells by confocal microscopy.
    Author: Nooh MM, Bahouth SW.
    Journal: Methods Cell Biol; 2017; 142():67-78. PubMed ID: 28964341.
    Abstract:
    G protein-coupled receptors (GPCRs) are recognized as one of the most fruitful group of therapeutic targets, accounting for more than 40% of all approved pharmaceuticals on the market. Therefore, the search for selective agents that affect GPCR function is of major interest to the pharmaceutical industry. This chapter describes methods for measuring agonist-promoted GPCR trafficking, which involves the internalization of the GPCR and its subsequent recycling back to the plasma membrane or retention and eventual degradation. These pathways will be analyzed by confocal cellular imaging, using the β1-adrenergic receptor (β1-AR) as a primary model. A major problem encountered in studying GPCR trafficking is the unavailability of antibodies that would recognize the native receptor in cells or tissues. Therefore, wild-type, point mutants, and β1-AR chimeras are generated as epitope-tagged proteins, which are stably- or transiently expressed in mammalian cells. GPCR are labeled with a fluorophore-conjugated antibody directed against the N-terminal epitope tag. The trafficking of the fluorophore-tagged GPCR between divergent trafficking pathways that result in retention and eventual degradation or recycling and reinsertion into the plasma membrane can be followed by confocal immunofluorescence microscopy techniques outlined in this review.
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