Molecular cloning and functional analysis of a novel P₂ nucleotide receptor

The cDNA encoding a novel P₂ receptor was isolated from rat aortic smooth muscle cell library and functionally characterized. The cloned P₂ receptor exhibits structural features characteristic of the G protein-coupled receptor family and shows 44 and 38% amino acid identity with previously cloned rat P(2U) and chicken P(2Y) receptors, respectively. The cloned P₂ receptor is functionally coupled to phospholipase C but not to adenylate cyclase in C6 rat glioma cells transfected with the cloned P₂ expression vector. The rank order of agonist potency as judged by intracellular Ca²⁺ mobilization responses is UTP > ADP = 2-methylthioATP > ADPβS > ATP = ATPγS, which is not compatible with any of the previously characterized P₂ receptor subtypes. The nonselective P₂ antagonists, suramin and reactive blue-2, inhibit nucleotide-induced phospholipase C activation in cells expressing the cloned P₂ receptor. The cloned P₂ receptor mRNA is abundantly expressed in various rat tissues including lung, stomach, intestine, spleen, mesentery, heart, and, most prominently, aorta. The results indicate that the novel metabotropic P₂ receptor has pharmacological characteristics distinct from any of P₂ receptor subtypes thus far identified and suggest the existence of a novel regulatory system by extracellular nucleotides of potential significance.