Cloning and functional expression of a human uridine nucleotide receptor

In order to isolate new subtypes of P-2 purinoceptors, sets of degenerate oligonucleotide primers were synthesized on the basis of the best conserved segments in the published sequences of the chick brain P-2Y/P2Y(1) and murine neuroblastoma P-2U/P2Y(2) receptors. Their use in polymerase chain reaction (PCR) experiments on human genomic DNA amplified, among other things, a 712-base pair sequence, that was used as a probe to screen a human genomic DNA library. Several clones corresponding to a single locus were isolated, and the sequence analysis revealed an intronless 1095-base pair open reading frame. The deduced amino acid sequence is consistent with a G protein-coupled receptor and exhibits 51% identity with the human P2Y(2) receptor and 35% with the chick P2Y(1) receptor. A close comparison with the human P2Y(2) sequence reveals the conservation of histidine 262, arginine 265, lysine 289, and arginine 292, which were reported to be involved in nucleotide binding (Erb, L., Garrad, R., Wang, Y., Quinn, T., Turner, J. T., and Weisman, G. A. (1995) J. Biol. Chem. 270, 4185-4188). Northern blot analysis detected a 1.8-kilobase messenger RNA in human placenta, The coding sequence was inserted in the pcDNA3 vector in order to transfect 1321N1 human astrocytoma cells. In cells stably expressing the receptor, UTP and UDP stimulated the formation of inositol phosphates with equivalent potency and maximal effect, ATP behaved as a partial agonist, and ADP was almost inactive. We have thus cloned a new member of the G protein coupled P-2 purinergic receptor family, which functionally behaves as a pyrimidinergic receptor.