Characterisation of a recombinant P2Y purinoceptor.
We have previously cloned a cDNA encoding a G-protein-coupled P2 purinoceptor from chick brain and designated this as a P2Y1 purinoceptor (Webb, T.E., J. Simon, B.J. Krishek, A.N. Bateson, T.G. Smart, B.J. King, G. Bumstock and E.A. Barnard, 1993, FEBS Lett. 324, 219). Here, we describe the further characterisation of this recombinant receptor expressed in both simian kidney endothelial (COS-7) cells and Xenopus oocytes. In transfected COS-7 cell membranes, the recombinant receptor showed a high level of expression (Bmax = 7.9 +/- 2.2. pmol [35S]dATP alpha S bound/mg protein) and affinity (Kd = 6.6 +/- 0.3 nM). In these COS-7 cells, the activation of the implanted purinoceptor induced a suramin-sensitive formation of inositol 1,4,5-triphosphatic (1,4,5InsP3). Upon expression in Xenopus oocytes, ATP was the only natural nucleoside triphosphate to elicit a Ca(2+)-activated chloride current. The P2 purinoceptor antagonists suramin and Reactive Blue-2 were both able to inhibit this evoked current. Utilizing both expression systems, the binding affinity profile and the functional pharmacological profile of the agonists, the common series found was: 2-methylthioATP (2-MeSATP) > or = ATP > ADP beta S > ADP. These two agonist series and the lack of activity of adenosine, alpha, beta-methyleneATP (alpha, beta-meATP), 3'-O-(4-benzoyl) benzoyl-ATP (Bz-ATP) and UTP, together confirmed that this receptor is a specific subtype of the P2Y purinoceptors.
Simon
J
J
Molecular Neurobiology Unit, Royal Free Hospital School of Mediìcine, Londan, UK.
Webb
T E
TE
King
B F
BF
Burnstock
G
G
Barnard
E A
EA
D000241:Adenosine / Q000737:chemistry; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry* / Q000494:pharmacology; D000345:Affinity Labels; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002460:Cell Line; D004594:Electrophysiology; D007295:Inositol Phosphates / Q000032:analysis; D018048:Receptors, Purinergic P2 / Q000096:biosynthesis / Q000737:chemistry* / Q000187:drug effects; D058472:Receptors, Purinergic P2Y1; D011994:Recombinant Proteins / Q000737:chemistry; D013498:Suramin / Q000737:chemistry; D013873:Thionucleotides / Q000737:chemistry; D014227:Triazines / Q000737:chemistry; D014544:Uridine Triphosphate / Q000737:chemistry; D014981:Xenopus
D000345:Affinity Labels; D007295:Inositol Phosphates; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D011994:Recombinant Proteins; D013873:Thionucleotides; D014227:Triazines; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; C002480:methylthio-ADP; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; C012842:Cibacron Blue F 3GA; D013498:Suramin; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000241:Adenosine; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; D014544:Uridine Triphosphate; C008501:2-methylthio-ATP