An examination of deoxyadenosine 5′(α‐thio)triphosphate as a ligand to define P2Y receptors and its selectivity as a low potency partial agonist of the P2Y1 receptor

The functional activity of deoxyadenosine 5′(α‐thio)triphosphate (dATPαS) was assessed at the cloned human P2Y1 receptor stably expressed in 1321N1 human astrocytoma cells and transiently expressed in Cos‐7 cells. Cells expressing the receptor responded to adenine nucleotides with an increase in [3H]‐inositol phosphate accumulation. Half‐maximal responses were obtained at approximately 30 nM for 2‐methylthioadenosine‐5′‐triphosphate (2MeSATP), 300 nM for dATPαS, and 1000 nM for adenosine 5′‐triphosphate (ATP). dATPαS produced a maximal response that was only 37±4% of that produced by ATP or 2MeSATP. dATPαS also competitively antagonized the phospholipase C response to 2MeSATP with a KB of 644±14 nM. Thus dATPαS acts as a low potency partial agonist at P2Y1 receptors. The selectivity of dATPαS for P2Y1 receptors was determined by examining its capacity to activate P2Y2, P2Y4 and P2Y6 receptors also stably expressed in 1321N1 cells. Although dATPαS was a partial agonist at P2Y1 receptors it was a full agonist at P2Y2 receptors, albeit with a potency that was two orders of magnitude lower than at P2Y1 receptors. No agonist or antagonist activity was observed at P2Y4 and P2Y6 receptors. Although [35S]‐dATPαS bound to a relatively high density (ca 10 pmol mg−1 protein) of binding sites in membranes from 1321N1 or Cos‐7 cells expressing the P2Y1 receptor, no difference in the total density of sites was observed between membranes from wild‐type, empty vector‐transfected, or P2Y1 receptor‐expressing cells. Moreover, adenine nucleotide analogues inhibited [35S]‐dATPαS binding with an order of potency that differed markedly from that for the accumulation of inositol phosphates in intact transfected P2Y1 receptor‐expressing cells. Saturation binding experiments demonstrated multiple affinity states for [35S]‐dATPαS binding in wild‐type Cos‐7 cell membranes. These data from 1321N1 and Cos‐7 cells suggest that cellular membranes exhibit a large number of high affinity binding sites for [35S]‐dATPαS that are not related to P2Y receptor subtypes. British Journal of Pharmacology (1997) 121, 338–344; doi:10.1038/sj.bjp.0701136