Agonist Binding and Gq-Stimulating Activities of the Purified Human P2Y₁Receptor

Abstract

The human P2Y 1 receptor (P2Y 1 -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 P2Y 1 -R antagonist [ binding assays for antagonists with the purified P2Y 1 -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 P2Y 1 -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) Ͼ ADP ϭ 2-methylthio-ATP ϭ adenosine-5Ј-O-(3-thio)triphosphate ϭ adenosine-5Ј-O-(2-thiodiphosphate) Ͼ Ͼ ATP was obtained. The signaling activity of the purified P2Y 1 -R was quantified after reconstitution in proteoliposomes with heterotrimeric G proteins. Steady-state GTP hydrolysis in vesicles reconstituted with P2Y 1 -R and G␣ q ␤ 1 ␥ 2 was stimulated by the addition of either 2MeADP or RGS4 alone and was increased by up to 50-fold in their combined presence. EC 50 values of agonists for activation of the purified P2Y 1 -R were similar to their respective K i values determined in radioligand binding experiments with the purified receptor. Moreover, ATP exhibited 20-fold higher EC 50 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 G␣ 11 in P2Y 1 -R-containing vesicles. These results illustrate that the binding and signaling properties of the human P2Y 1 -R can be studied with purified proteins under conditions that circumvent the complications that occur in vivo.Burnstock's hypothesis (Burnstock, 1972) of purinergic signaling largely has been confirmed over the past two decades by demonstration of regulated release of cellular nucleotides, by elucidation of the mechanisms of metabolism of extracellular nucleotides by a complex array of ecto-enzymes, and by delineation of myriad nucleotide-promoted physiological responses (Dubyak and El-Moatassim, 1993;Harden et al., 1995;Ralevic and Burnstock, 1998;Lazarowski et al., 2003). Moreover, the molecular cloning of two classes of ubiquitously distributed receptors for extracellular nucleotides, the ligand-gated P2X receptors (Khakh et al., 2000) and the G protein-coupled P2Y receptors , comprising at least 15 human genes, has amplified the importance of nucleotide-promoted signaling.Although a burgeoning interest in the biology and therapeutic potential of extracellular nucleotide signaling now exists, the complex regulatory pathways that underlie the action of extracellular nucleotides remain difficult to study. Few truly selective agonists and antagonists are available for the P2 receptors, and studies with intact tissues are compromised both by nucleotide release and by metabolism and interconversion of ...

A molecular signaling model of platelet phosphoinositide and calcium regulation during homeostasis and P2Y1 activation

“…We have focused on the subset of reactions that govern ADP-mediated phosphoinositide signaling and mobilization of intracellular Ca 2ϩ ( Table 1). The P2Y 1 receptor is coupled primarily to the G q family of G proteins, 20,21 which activate the beta-isoform of phospholipase C. 1,20 Previous work has shown that this pathway accounts for more than 90% of the ADP-mediated Ca 2ϩ signaling in platelets. 22 To eliminate Ca 2ϩ influx and the need to model store-operated Ca 2ϩ entry, we used experimental data from studies in which extracellular Ca 2ϩ was removed by ethylenediaminetetraacetic acid (EDTA) or another chelating agent.…”

“…In that assay, Waldo and Harden combined purified human P2Y 1 , ADP, G ␣ and G ␤␥ subunits, PLC-␤ or RGS4, and radiolabeled GTP in large phospholipid vesicles and measured the agonist binding and G q -stimulating activities of the receptor. 21 GTPase activity was measured with and without agonist ( Figure 4B). ADP dose-response was measured with and without GAP protein ( Figure 4C).…”

Investigation of the functional expression of purine and pyrimidine receptors in porcine isolated pancreatic arteries

“…Molecular cloning has identified seven mammalian P2X receptor subunits: P2X1, P2X2, P2X3, P2X4, P2X5, P2X6 and P2X7 [3], while eight mammalian P2Y receptors have been identified: P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , P2Y 13 and P2Y 14 [4]. P2X receptors are activated by adenosine-5′-triphosphate (ATP) and its stable, and consequently more potent, analogue αβ-methylene-ATP (αβ-meATP) [5,6].…”

“…P2X receptors are activated by adenosine-5′-triphosphate (ATP) and its stable, and consequently more potent, analogue αβ-methylene-ATP (αβ-meATP) [5,6]. P2Y receptors can be divided on the basis of their endogenous agonists into adenine nucleotide-preferring (P2Y 1 , P2Y 11 , P2Y 12 and P2Y 13 ) receptors and uracil nucleotide or UDP-sugar-preferring (P2Y 2 , P2Y 4 , P2Y 6 and P2Y 14 ) receptors [7]. Among the adenine nucleotide group, the human P2Y 11 receptor is selectively activated by ATP and fails to respond to adenosine-5′-diphosphate (ADP) [8], although the dog orthologue responds to both ADP and ATP [9].…”

“…Among the adenine nucleotide group, the human P2Y 11 receptor is selectively activated by ATP and fails to respond to adenosine-5′-diphosphate (ADP) [8], although the dog orthologue responds to both ADP and ATP [9]. P2Y 1 , P2Y 12 and P2Y 13 receptors are activated by ADP and, with lower potency, by ATP [10][11][12][13]. Among the uracil nucleotide or UDP-sugar receptors, the P2Y 2 receptor is equally activated by ATP and uridine-5′-triphosphate (UTP), while the P2Y 4 receptor is highly selective for UTP over ATP [14].…”