Novel modified adenosine 5 '-triphosphate analogues pharmacologically characterized in human embryonic kidney 293 cells highly expressing rat brain P2Y(1) receptor: biotinylated analogue potentially suitable for specific P2Y(1) receptor isolation

Rat brain P2Y(1) (rP2Y(1)) receptor-transfected human embryonic kidney cell s (HEK 293) were recently shown to have enhanced reactivity to both ATP and ADP (Vohringer C, Schafer R, Reiser G. Biochem Pharmacol 2000;59:791-800). Here, we demonstrated the usefulness of this cell line as a system for fur ther studying novel adenine nucleotide analogues (Halbfinger et al. J Med C hem 1999;42:5325-37) and for the biochemical characterization of the P2Y(1) receptor. By measurement of intracellular Ca2+ release, for 2-butylthio-, 2-butylamino-, and 2-butyloxy-ATP (2-BuS-, 2-BuNH-, 2-BuO-ATP), EC50 values of 1.3, 5, and 60 nM were determined, markedly lower than the value for AT P (130 nM). The EC50 for 2-BuSADP was 1.1 nM. The corresponding 8-substitut ed ATP analogues showed a substantially lower potency than ATP (ATP > 8-BuS ATP > 8-BuNHATP approximate to 8-BuOATP). AMP induced intracellular Ca2+ re lease with a very low potency; 2- and 8-substitutions on AMP caused no sign ificant potency shift, except for 2-BuSAMP (EC50 = 180 nM). Another new P2Y receptor probe, 2-[(6-biotinylamido)-hexylthio]ATP, was 22-fold more poten t than ATP (EC50 = 6 nM). revealing that even more bulky substituents linke d to the C-2 position bind with high affinity at the P2Y(1) receptor. This biotinylated probe was successfully used for the enrichment of the P2Y(1) r eceptor tagged with green fluorescent protein from a crude membrane fractio n. This one-step enrichment provides a substantial advance for P2Y(1) recep tor purification. Thus, human embryonic kidney 293 cells stably transfected with the rP2Y(1) receptor represent a powerful model system for pharmacolo gical characterization of the P2Y(1) receptor, circumventing problems assoc iated with natural systems. They provide a means for the development of P2Y (1) ligands of high potency and a good source for obtaining purified P2Y(1) receptor. (C) 2001 Elsevier Science Inc. All rights reserved.