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Abstract
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.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X91852
We describe the cloning of a new member of the P2Y family, tentatively called P2Y4, which exhibits a preference for uridine over adenine nucleotides and constitutes the first example of a pyrimidinergic receptor
Cloning and Sequencing—In order to isolate new subtypes of P2 receptors, sets of degenerate oligonucleotides primers were synthesized on the basis of the best conserved segments in the published sequences of the chick brain P2Y1 [5] and murine neuroblastoma P2Y2 [9] receptors
Summary
(Received for publication, August 31, 1995, and in revised form, October 12, 1995). From the Institute of Interdisciplinary Research, School of Medicine, and the ʈDepartment of Medical Chemistry, Erasme Hospital, Universite Libre de Bruxelles, 1070 Brussels, Belgium. 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 712base pair sequence, that was used as a probe to screen a human genomic DNA library. We describe the cloning of a new member of the P2Y family, tentatively called P2Y4, which exhibits a preference for uridine over adenine nucleotides and constitutes the first example of a pyrimidinergic receptor
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