TY - JOUR T1 - Agonist Binding and Gq-Stimulating Activities of the Purified Human P2Y<sub>1</sub> Receptor JF - Molecular Pharmacology JO - Mol Pharmacol SP - 426 LP - 436 DO - 10.1124/mol.65.2.426 VL - 65 IS - 2 AU - Gary L. Waldo AU - T. Kendall Harden Y1 - 2004/02/01 UR - http://molpharm.aspetjournals.org/content/65/2/426.abstract N2 - The human P2Y1 receptor (P2Y1-R) was purified after high-level expression from a recombinant baculovirus in Sf9 insect cells. Quantification by protein staining and with a radioligand binding assay using the high-affinity P2Y1-R antagonist [3H]MRS2279 ([3H]2-chloro-N6-methyl-(N)-methanocarba-2′-deoxyadenosine 3′,5′-bis-phosphate) indicated a nearly homogenous preparation of receptor protein. Ki values determined in [3H]MRS2279 binding assays for antagonists with the purified P2Y1-R were in good agreement with the Ki and KB values determined for these molecules in membrane binding and activity assays, respectively. Availability of P2Y1-R in purified form allowed direct determination of nucleotide agonist affinities under conditions not compromised by nucleotide metabolism/interconversion, and an order of affinities of 2-methylthio-ADP (2MeSADP) &gt; ADP = 2-methylthioATP = adenosine-5′-O-(3-thio)triphosphate = adenosine-5′-O(2-thiodiphosphate) ≫ ATP was obtained. The signaling activity of the purified P2Y1-R was quantified after reconstitution in proteoliposomes with heterotrimeric G proteins. Steady-state GTP hydrolysis in vesicles reconstituted with P2Y1-R and Gαqβ1γ2 was stimulated by the addition of either 2MeADP or RGS4 alone and was increased by up to 50-fold in their combined presence. EC50 values of agonists for activation of the purified P2Y1-R were similar to their respective Ki values determined in radioligand binding experiments with the purified receptor. Moreover, ATP exhibited 20-fold higher EC50 and Ki values than did ADP and was a partial agonist relative to ADP and 2MeSADP under conditions in which no metabolism of the nucleotide occurred. Both RGS4 and PLC-β1 were potent and efficacious GTPase-activating proteins for Gαq and Gα11 in P2Y1-R–containing vesicles. These results illustrate that the binding and signaling properties of the human P2Y1-R can be studied with purified proteins under conditions that circumvent the complications that occur in vivo. ER -