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Purification and Functional Reconstitution of the Human P2Y₁₂ Receptor

Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina

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, G₁₂, and various G-subunits were reconstituted in lipid vesicles, and steady-state GTPase activity was quantified. GTP hydrolysis in proteoliposomes formed with purified P2Y₁₂-R and G_i212 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 -subunits in heterotrimeric form with G₁₂. The most robust coupling of the P2Y₁₂-R was to G_i2, but effective coupling also occurred to G_i1 and G_i3. In contrast, little or no coupling occurred to G_o or G_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.

Received July 15, 2003; accepted August 13, 2003.

Address correspondence to: Dr. T. Kendall Harden, Department of Pharmacology, CB#7365, University of North Carolina Chapel Hill, Chapel Hill, NC 27599-7365. E-mail: tkh{at}med.unc.edu

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