Tandem Affinity Purification and Identification of GPCR-Associated Protein Complexes

International audienceThe first tandem affinity purification (TAP) protocol was described in 1999. Originally designed for the purification of protein complexes in yeast RNA splicing, its application rapidly expanded towards whole proteome analysis in yeast and mammalian cells. More recently, TAP has been applied to the purification of G protein-coupled receptor (GPCR)-associated protein complexes (GAPCs). This approach is particularly attractive for GPCRs, as the native, seven transmembrane structure is used as bait to purify GAPCs from mammalian cells expressing receptors at physiological levels. Here, a detailed protocol of the TAP method applied to GPCRs is presented. Members of the G protein-coupled receptor (GPCR) superfamily share a common and complex topology consisting of an extracellu-lar amino-terminal domain, a hydrophobic core of seven trans-membrane (7TM) a-helices that interact together to form a three-dimensional barrel within the plasma membrane, and a cyto-solic carboxyl terminus (C-terminus) (1). Whereas extracellular loops and the hydrophobic 7TM core are involved in ligand binding , the intracellular domain of the receptor, composed of three loops and the C-terminus, are important for signal transmission, receptor trafficking and desensitization. All these functions are accompanied by the dynamic recruitment of different protein complexes to intracellular receptor subdomains. Some interactions rely on linear molecular determinants of one single receptor subdomain