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 P2Y(1) RECEPTOR

1 The functional activity of deoxyadenosine 5'(alpha-thio)triphosphate (dATP alpha S) was assessed at the cloned human P2Y(1) receptor stabl y expressed in 1321N1 human astrocytoma cells and transiently expresse d in Cos-7 cells. 2 Cells expressing the receptor responded to adenine nucleotides with an increase in [H-3]-inositol phosphate accumulation . Half-maximal responses were obtained at approximately 30 nM for 2-me thylthioadenosine-5'-triphosphate (2MeSATP), 300 nM for dATP alpha S, and 1000 nM for adenosine 5'-triphoshate (ATP). dATP alpha S produced a maximal response that was only 37+/-4% of that produced by ATP or 2M eSATP. dATP alpha S also competitively antagonized the phospholipase C response to 2MeSATP with a K-B of 644+/-14 nM. Thus dATP alpha S acts as a low potency partial agonist at P2Y(1) receptors, 3 The selectivi ty of dATP alpha S for P2Y(1) receptors was determined by examining it s capacity to activate P2Y(2), P2Y(4) and P2Y(6) receptors also stably expressed in 1321N1 cells. Although dATP alpha S was a partial agonis t at P2Y(1) receptors it was a full agonist al P2Y(2) receptors, albei t with a potency that was two orders of magnitude lower than at P2Y(1) receptors; No agonist or antagonist activity was observed at P2Y(4) a nd P2Y(6) receptors. 4 Although [S-35]-dATP alpha S bound to a relativ ely high density (ca 10 pmol mg(-1) protein) of binding sites in membr anes from 1321N1 or Cos-7 cells expressing the P2Y(1) receptor, no dif ference in the total density of sites was observed between membranes f rom wild-type empty vector-transfected, or P2Y(1) receptor-expressing cells, Moreover, adenine nucleotide analogues inhibited [S-35]-dATP al pha S binding with an order of potency that differed markedly from tha t for the accumulation of inositol phosphates in intact transfected P2 Y(1) receptor-expressing cells. Saturation binding experiments demonst rated multiple affinity stales for [S-35]-dATP alpha S binding in wild -type Cos-7 cell membranes, These data from 1321N1 and Cos-7 cells sug gest that cellular membranes exhibit a large number of high affinity b inding sites for [S-35]-dATP alpha S that are not related to P2Y recep tor subtypes.