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Agonist Binding and Gq-Stimulating Activities of the Purified Human P2Y₁ Receptor

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

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. K_i values determined in [³H]MRS2279 binding assays for antagonists with the purified P2Y₁-R were in good agreement with the K_i and K_B 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) > 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 G_q12 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 K_i values determined in radioligand binding experiments with the purified receptor. Moreover, ATP exhibited 20-fold higher EC₅₀ and K_i 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-1 were potent and efficacious GTPase-activating proteins for G_q and G₁₁ 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.

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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