Cloning and characterization of Xenopus P2Y receptors

Adenosine 5'-triphosphate (ATP) is co-stored with acetylcholine (ACh) in the synaptic vesicles and co-released to muscle fiber upon muscle contraction. From previous study, P2Y receptors were detected as dominant ATP receptors on chick muscle. However, the exact role of ATP and its receptors in the formation of neuromuscular junction is still unknown. In order to study their possible roles, I have cloned and characterized P2Y receptors from Xenopus embryonic cDNA library. By using chick P2Y1 receptor cDNA as a probe, we isolated Xenopus P2Y1 receptor, which shares 70%-90% amino acid homology with other species. In [35S] dATPαS competitive ligand binding assay, the recombinant Xenopus P2Y1 receptor showed a strong affinity to P2Y1 agonists, like 2-MeSADP and ATP. Application of P2Y1 receptor agonists on the recombinant Xenopus P2Y1 receptor could stimulate the induction of AChR α-subunit and AChE promoter activities in chick myotubes. In situ hybridization showed a strong expression of Xenopus P2Y1 receptor in the brain, spinal cord and myotomes at developing Xenopus embryo. By using immunohistochemical analysis, P2Y1, P2Y2 and P2Y4 receptors were co-localized with AChRs at Xenopus neuromuscular junction. In addition, a partial clone related to P2Y2 receptor was isolated and found to have high amino acid homology to other members of P2Y2 receptor. These evidences suggest the possible role of ATP and P2Y receptors in the formation of neuromuscular junction