title: 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.
abstract: Rat brain P2Y(1) (rP2Y(1)) 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 P2Y(1) receptor. By measurement of intracellular Ca(2+) release, for 2-butylthio-, 2-butylamino-, and 2-butyloxy-ATP (2-BuS-, 2-BuNH-, 2-BuO-ATP), EC(50) values of 1.3, 5, and 60 nM were determined, markedly lower than the value for ATP (130 nM). The EC(50) 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 approximately 8-BuOATP). AMP induced intracellular Ca(2+) release with a very low potency; 2- and 8-substitutions on AMP caused no significant potency shift, except for 2-BuSAMP (EC(50) = 180 nM). Another new P2Y receptor probe, 2-[(6-biotinylamido)-hexylthio]ATP, was 22-fold more potent than ATP (EC(50) = 6 nM), revealing that even more bulky substituents linked 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) receptor tagged with green fluorescent protein from a crude membrane fraction. This one-step enrichment provides a substantial advance for P2Y(1) receptor purification. Thus, human embryonic kidney 293 cells stably transfected with the rP2Y(1) receptor represent a powerful model system for pharmacological characterization of the P2Y(1) receptor, circumventing problems associated 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.
authors.affiliation: Institut für Neurobiochemie, Otto-von-Guericke-Universität, Medizinische Fakultät, Leipziger Strasse 44, D-39120, Magdeburg, Germany.
authors.forename: G
authors.initials: G
authors.lastname: Zündorf
authors.forename: R
authors.initials: R
authors.lastname: Schäfer
authors.forename: C
authors.initials: C
authors.lastname: Vöhringer
authors.forename: E
authors.initials: E
authors.lastname: Halbfinger
authors.forename: B
authors.initials: B
authors.lastname: Fischer
authors.forename: G
authors.initials: G
authors.lastname: Reiser
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology*; D000818:Animals; D019921:Biotinylation; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology; D058910:Purinergic P2 Receptor Agonists; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000302:isolation & purification*; D058472:Receptors, Purinergic P2Y1; D013329:Structure-Activity Relationship; D013873:Thionucleotides / Q000737:chemistry / Q000494:pharmacology*
chemical_list: C546382:P2RY1 protein, human; C546384:P2ry1 protein, rat; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D013873:Thionucleotides; D000255:Adenosine Triphosphate; D002118:Calcium; C008501:2-methylthio-ATP