Purification and Functional Reconstitution of the Human P2Y12 Receptor

The human P2Y12 receptor (P2Y12-R) is a member of the G protein coupled P2Y receptor family, which is intimately in-volved in platelet physiology. We describe here the purification and functional characterization of recombinant P2Y12-R after high-level expression from a baculovirus in Sf9 insect cells. Purified P2Y12-R, G12, and various G-subunits were recon-stituted in lipid vesicles, and steady-state GTPase activity was quantified. GTP hydrolysis in proteoliposomes formed with pu-rified P2Y12-R and Gi212 was stimulated by addition of either 2-methylthio-ADP (2MeSADP) or RGS4 and was mark-edly enhanced by their combined presence. 2MeSADP was the most potent agonist (EC50 80 nM) examined, whereas ADP, the cognate agonist of the P2Y12-R, was 3 orders of magnitude less potent. ATP had no effect alone but inhibited the action o