CHARACTERIZATION OF A RECOMBINANT P-2Y PURINOCEPTOR

We have previously cloned a cDNA encoding a G-protein-coupled P-2 puri noceptor from chick brain and designated this as a P-2Y1 purinoceptor (Webb, T.E., J. Simon, B.J. Krishek, A.N. Bateson, T.G. Smart, B.J. Ki ng, G. Burnstock and E.A. Barnard, 1993, FEES Lett. 324, 219). Here, w e describe the further characterisation of this recombinant receptor e xpressed in both simian kidney endothelial (COS-7) cells and Xenopus o ocytes. In transfected COS-7 cell membranes, the recombinant receptor showed a high level of expression (B-max = -7.9 +/- 2.2 pmol [S-35]dAT P alpha S bound/mg protein) and affinity (K-d = 6.6 +/- 0.3 nM). In th ese COS-7 cells, the activation of the implanted purinoceptor induced a suramin-sensitive formation of inositol 1,4,5-trisphosphate (1,4,5In sP(3)). Upon expression in Xenopus oocytes, ATP was the only natural n ucleoside triphosphate to elicit a Ca2+-activated chloride current. Th e P-2 purinoceptor antagonists suramin and Reactive Blue-2 were both a ble to inhibit this evoked current. Utilizing both expression systems, the binding affinity profile and the functional pharmacological profi le of the agonists, the common series found was: 2-methylthioATP (2-Me SATP) greater than or equal to ATP > ADP beta S > ADP. These two agoni st series and the lack of activity of adenosine, alpha,beta-methyleneA TP (alpha,beta-meATP), 3'-O-(4-benzoyl)benzoyl-ATP (Bz-ATP) and UTP, t ogether confirmed that this receptor is a specific subtype of the P-2Y purinoceptors.