Abstract

The human P2Y₁₂ receptor (P2Y₁₂-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 P2Y₁₂-R after high-level expression from a baculovirus in Sf9 insect cells. Purified P2Y₁₂-R, Gβ₁γ₂, and various Gα-subunits were reconstituted in lipid vesicles, and steady-state GTPase activity was quantified. GTP hydrolysis in proteoliposomes formed with purified P2Y₁₂-R and Gα_i2β₁γ₂ 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 P2Y₁₂-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 P2Y₁₂-R. The G protein selectivity of the P2Y₁₂-R was examined by reconstitution with various G protein α-subunits in heterotrimeric form with Gβ₁γ₂. The most robust coupling of the P2Y₁₂-R was to Gα_i2, but effective coupling also occurred to Gα_i1 and Gα_i3. In contrast, little or no coupling occurred to Gα_o or Gα_q. These results illustrate that the signaling properties of the P2Y₁₂-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.