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 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, Gbeta1gamma2, and various Galpha-subunits were reconstituted in lipid vesicles, and steady-state GTPase activity was quantified. GTP hydrolysis in proteoliposomes formed with purified P2Y12-R and Galphai2beta1gamma2 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 alpha-subunits in heterotrimeric form with Gbeta1gamma2. The most robust coupling of the P2Y12-R was to Galphai2, but effective coupling also occurred to Galphai1 and Galphai3. In contrast, little or no coupling occurred to Galphao or Galphaq. 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.
Department of Pharmacology, University of North Carolina Chapel Hill, Chapel Hill, NC 27599-7365, USA.
Erik T
ET
Bodor
Gary L
GL
Waldo
Shelley B
SB
Hooks
James
J
Corbitt
Jose L
JL
Boyer
T Kendall
TK
Harden
D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D006801:Humans; D008565:Membrane Proteins; D018048:Receptors, Purinergic P2 / Q000302:isolation & purification* / Q000502:physiology; D058925:Receptors, Purinergic P2Y12; D014162:Transfection
D008565:Membrane Proteins; C548576:P2RY12 protein, human; D018048:Receptors, Purinergic P2; D058925:Receptors, Purinergic P2Y12; D000244:Adenosine Diphosphate