In order to isolate new subtypes of
P2 purinoceptors, sets of degenerate
oligonucleotide primers were synthesized on the basis of the best conserved segments in the published sequences of the chick brain P2Y/P2Y1 and murine
neuroblastoma P2U/
P2Y2 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
P2Y2 receptor and 35% with the chick
P2Y1 receptor. A close comparison with the human P2Y2 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 P2
purinergic receptor family, which functionally behaves as a pyrimidinergic receptor.