Rat brain P2Y1 (rP2Y1) receptor-transfected human embryonic kidney cells (HEK 293) were recently shown to have enhanced reactivity to both ATP and ADP (Vöhringer C, Schäfer R, Reiser G. Biochem Pharmacol 2000;59:791-800). Here, we demonstrated the usefulness of this cell line as a system for further studying novel adenine nucleotide analogues (Halbfinger et al. J Med Chem 1999;42:5325-37) and for the biochemical characterization of the P2Y1 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 ATP (130 nM). The EC50 for 2-BuSADP was 1.1 nM. The corresponding 8-substituted ATP analogues showed a substantially lower potency than ATP (ATP > 8-BuSATP > 8-BuNHATP ≈ 8-BuOATP). AMP induced intracellular Ca2+ release with a very low potency; 2- and 8-substitutions on AMP caused no significant potency shift, except for 2-BuSAMP (EC50 = 180 nM). Another new P2Y receptor probe, 2-[(6-biotinylamido)-hexylthio]ATP, was 22-fold more potent than ATP (EC50 = 6 nM), revealing that even more bulky substituents linked to the C-2 position bind with high affinity at the P2Y1 receptor. This biotinylated probe was successfully used for the enrichment of the P2Y1 receptor tagged with green fluorescent protein from a crude membrane fraction. This one-step enrichment provides a substantial advance for P2Y1 receptor purification. Thus, human embryonic kidney 293 cells stably transfected with the rP2Y1 receptor represent a powerful model system for pharmacological characterization of the P2Y1 receptor, circumventing problems associated with natural systems. They provide a means for the development of P2Y1 ligands of high potency and a good source for obtaining purified P2Y1 receptor.
Bibliographical noteFunding Information:
The work was supported by Graduiertenkolleg of Deutsche Forschungsgemeinschaft (GRK253), Fonds der Chemischen Industrie (G.R.), and the German-Israeli-Foundation (I0513-210.09/96; G.R. and B.F.). This work was also supported in part by the Marcus Center for Medicinal Chemistry (B.F.). We thank Dr. J. Ubl for helpful advice and K. Christoph for expert technical assistance with the cell cultures.
- Adenine nucleotides
- P2Y receptor ligands
- Purinergic receptor
- Receptor expression