[32P]2-iodo-N6-methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate ([32P]MRS2500), a novel radioligand for quantification of native P2Y1 receptors

1 Analysis of the P2Y family of nucleotide-activated G-protein-coupled receptors has been compromised by the lack of selective high-affinity, high-specific-radioactivity radioligands. We have pursued quantification of the P2Y(1) receptor through the development of a series of selective P2Y(1) receptor antagonists. 2 Recently, we synthesized 2-iodo-N-6-methyl-(N)- methanocarba-2'-deoxyadenosine 3',5'-bisphosphate (MRS2500), a selective, competitive antagonist that exhibits a K-i of 0.8 nM in competition-binding assays with [H-3] MRS2279. A 3'- monophosphate precursor molecule, MRS2608, was radiolabeled at the 5' position with P-32 using polynucleotide kinase and [gamma P-32] ATP to yield [P-32] MRS2500. 3 [P-32] MRS2500 bound selectively to Sf9 insect cell membranes expressing the human P2Y(1) receptor (Sf9-P2Y(1)), but did not detectably bind membranes expressing other P2Y receptors. P2Y(1) receptor binding to [P-32] MRS2500 was saturable with a K-D of 1.2 nM. Agonists and antagonists of the P2Y(1) receptor inhibited [P-32] MRS2500 binding in Sf9-P2Y(1) membranes with values in agreement with those observed in functional assays of the P2Y(1) receptor. 4 A high-affinity binding site for [P-32] MRS2500 (K-D 0.33 nM) was identified in rat brain, which exhibited the pharmacological selectivity of the P2Y(1) receptor. Distribution of this binding site varied among rat tissues, with the highest amount of binding appearing in lung, liver, and brain. Among brain regions, distribution of the [P-32] MRS2500 binding site varied by six-fold, with the highest and lowest amounts of sites detected in cerebellum and cortex, respectively. 5 Taken together, these data illustrate the synthesis and characterization of a novel P2Y(1) receptor radioligand and its utility for examining P2Y(1) receptor expression in native mammalian tissues.