Second Messenger Cascade Specificity and Pharmacological Selectivity of the Human P2Y1-purinoceptor

1. The coding sequence of the P2Y1-purinoceptor was cloned from a human genomic library. 2. The open reading frame encodes a protein of 373 amino acids that is 83% identical to the previously cloned chick and turkey P2Y1-purinoceptor and is > or = 95% homologous to the recently cloned rat, mouse, and bovine P2Y1-purinoceptors. 3. The human P2Y1-purinoceptor was stably expressed in 1321N1 human astrocytoma cells using a retroviral vector. Although the P2Y1-purinoceptor agonist, 2MeSATP, had no effect on inositol phosphate accumulation in cells infected with the P2Y1-purinoceptor virus. No effect of 2MeSATP on cyclic AMP accumulation was observed in P2Y1-receptor-expressing 1321N1 cells. 4. The pharmacological selectively of 18 purinoceptor agonists was established for the expressed human P2Y1-purinoceptor. 2MeSATp was more potent than ATP but less potent than 2MeSADP. ADP also was more potent than ATP. A similar maximal effect was observed with most agonists tested. However, alpha, beta-MeATP had no effect and 3'-NH2-3'-deoxyATP and A2P4 were partial agonists. The order of potency of agonists for activation of the turkey P2Y1-purinoceptor, also stably expressed in 1321N1 cells, was identical to that observed for the human P2Y1-purinoceptor. 5. C6 glioma cells express a P2Y-purinoceptor that inhibits adenylyl cyclase but does not activate phospholipase C. Expression of the human P2Y1-purinoceptor in C6 cells conferred 2MeSATP-stimulated inositol lipid hydrolysis to these cells. The phospholipase C-activating human P2Y1-purinoceptor could be delineated from the endogenous P2Y-purinoceptor of C6 glioma cells by use of the P2-purinoceptor antagonist, PPADS, which blocks the P2Y1-purinoceptor but does not block the endogenous P2Y-purinoceptor of C6 cells. P2-purinoceptor agonists also exhibited differential selectivities for activation of these two P2Y-purinoceptors.