Abstract
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) > 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.
- Received September 3, 2003.
- Accepted November 5, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|