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

Abstract

1. 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.

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 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 K_B of 644±14 nM. Thus dATPαS acts as a low potency partial agonist at P2Y₁ receptors.

3. 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.

4. 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.

Full Text

The Full Text of this article is available as a PDF (388.9 KB).