EXPRESSION OF A CLONED P-2Y PURINERGIC RECEPTOR THAT COUPLES TO PHOSPHOLIPASE-C

P-2Y purinergic receptors previously have been shown to couple either to activation of phospholipase C through a pertussis toxin-insensitive mechanism or to inhibition of adenylyl cyclase through pertussis toxi n-sensitive members of the G(i) family of G proteins. These and other pharmacological data strongly suggest that multiple P-2Y purinergic re ceptors exist. Webb et al. [FEBS Lett. 324:219-225 (1993)] cloned a cD NA that, when expressed in frog oocytes, displayed the general pharmac ological characteristics of a P-2Y purinergic receptor but whose secon d messenger linkage was not resolved. We have now cloned the meleagrid (turkey) homologue of the previously cloned chick P-2Y purinergic rec eptor and have stably expressed it in a heterologous human cell line ( 1321N1 astrocytoma cells) to establish its signaling properties. The p urinergic receptor agonist 2-methylthio-ATP (2MeSATP) stimulated a mar ked activation of phospholipase C in 1321N1 cells stably expressing th e meleagrid receptor. The order of potency of a series of analogues of ATP and ADP for stimulation of phospholipase C by the receptor expres sed in 1321N1 cells [2MeSATP = 2-methylthio-ADP > adenosine 5'O-(2-thi o)diphosphate > ADP > 2-chloro-ATP = adenosine 5'O-(3-thio)triphosphat e greater than or equal to ATP > adenylyl-imidodiphosphate > UTP] was similar to that observed for P-2Y purinergic receptors in turkey eryth rocytes and many other tissues and was markedly different from those o f the P-2U and P-2X purinergic receptor subtypes. Stimulation of inosi tol lipid hydrolysis by P-2Y purinergic agonists was not affected by p reincubation of cells with pertussis toxin. In contrast to its marked effects on phospholipase C activity, 2MeSATP caused only a small and v ariable inhibition of cAMP accumulation. Ribonuclease protection analy sis of turkey tissues showed that this P-2Y purinergic receptor is mos t highly expressed in blood and brain. Taken together, these results i ndicate that a phospholipase-C-activating P-2Y purinergic receptor has been cloned and stably expressed in 1321N1 astrocytoma cells.