Purification and functional reconstitution of the human P2Y12 receptor

The human P2Y(12) receptor (P2Y(12)-R) is a member of the G protein coupled P2Y receptor family, which is intimately involved in platelet physiology. We describe here the purification and functional characterization of recombinant P2Y(12)-R after high-level expression from a baculovirus in Sf9 insect cells. Purified P2Y(12)-R, Gbeta(1)gamma(2), and various Galpha-subunits were reconstituted in lipid vesicles, and steady-state GTPase activity was quantified. GTP hydrolysis in proteoliposomes formed with purified P2Y(12)-R and Galpha(i2)beta(1)gamma(2) was stimulated by addition of either 2-methylthio-ADP (2MeSADP) or RGS4 and was markedly enhanced by their combined presence. 2MeSADP was the most potent agonist (EC50 = 80 nM) examined, whereas ADP, the cognate agonist of the P2Y(12)-R, was 3 orders of magnitude less potent. ATP had no effect alone but inhibited the action of 2MeSADP; therefore, ATP is a relatively low-affinity antagonist of the P2Y(12)-R. The G protein selectivity of the P2Y(12)-R was examined by reconstitution with various G protein alpha-subunits in heterotrimeric form with Gbeta(1)gamma(2). The most robust coupling of the P2Y(12)-R was to Galpha(i2), but effective coupling also occurred to Galpha(i1) and Galpha(i3). In contrast, little or no coupling occurred to Galpha(o) or Galpha(q). These results illustrate that the signaling properties of the P2Y(12)-R can be studied as a purified protein under conditions that circumvent the complications that occur in vivo because of nucleotide metabolism and interconversion as well as nucleotide release.