Second messenger cascade specificity and pharmacological selectivity of the human P2Y1‐purinoceptor

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Abstract
. The coding sequence of the P2Y1‐purinoceptor was cloned from a human genomic library. . 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 ≥95% homologous to the recently cloned rat, mouse, and bovine P2Y1‐purinoceptors. . 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 1321N1 cells infected with the control virus, this agonist markedly stimulated 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. . The pharmacological selectivity 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, α,β‐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. . 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.