title: An examination of deoxyadenosine 5'(alpha-thio)triphosphate as a ligand to define P2Y receptors and its selectivity as a low potency partial agonist of the P2Y1 receptor.
abstract: 1. The functional activity of deoxyadenosine 5'(alpha-thio)triphosphate (dATP alpha S) was assessed at the cloned human P2Y1 receptor stably expressed in 1321N1 human astrocytoma cells and transiently expressed in Cos-7 cells. 2. 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 dATP alpha S, and 1000 nM for adenosine 5'-triphosphate (ATP). dATP alpha S produced a maximal response that was only 37 +/- 4% of that produced by ATP or 2MeSATP. dATP alpha S also competitively antagonized the phospholipase C response to 2MeSATP with a KB of 644 +/- 14 nM. Thus dATP alpha S acts as a low potency partial agonist at P2Y1 receptors. 3. The selectivity of dATP alpha S for P2Y1 receptors was determined by examining its capacity to activate P2Y2, P2Y4 and P2Y6 receptors also stably expressed in 1321N1 cells. Although dATP alpha S 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. 4. Although [35S]-dATP alpha S 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]-dATP alpha S 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]-dATP alpha S 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]-dATP alpha S that are not related to P2Y receptor subtypes.
authors.affiliation: Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill 27599-7365, USA.
authors.forename: J B
authors.initials: JB
authors.lastname: Schachter
authors.forename: T K
authors.initials: TK
authors.lastname: Harden
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D011869:Radioligand Assay; D018048:Receptors, Purinergic P2 / Q000187:drug effects*; D058472:Receptors, Purinergic P2Y1
chemical_list: C546382:P2RY1 protein, human; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D000255:Adenosine Triphosphate