Irreversible activation of phospholipase C-coupled P2Y-purinergic receptors by 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate

Abstract

3'-O-(4-Benzoyl)benzoyladenosine 5'-triphosphate (BzATP), a photoaffinity analog of ATP, was used as a ligand for the phospholipase C-linked P2Y-purinergic receptor in turkey erythrocytes. In membranes from [3H]inositol-labeled turkey erythrocytes, BzATP stimulated inositol phosphate formation in a concentration-dependent manner (K0.5 = 172 +/- 4 nM). The effect of BzATP was strictly dependent on the presence of guanine nucleotides and was not additive with the effect of other full P2Y-purinergic receptor agonists. Photolysis of [3H]-inositol-labeled membranes in the presence of BzATP had no effect on the magnitude of the maximally attainable response of phospholipase C to the P2Y-purinergic receptor agonist adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) plus guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). However, the effects of submaximally effective concentrations of GTP gamma S were markedly increased in membranes previously photolyzed in the presence of BzATP. In addition, the rate of activation of phospholipase C by GTP gamma S in membranes photolyzed in the presence of BzATP was increased 3-fold, as compared with control membranes. BzATP effects on phospholipase C were prevented by photolysis in the presence of ATP or ADP, but not by the presence of the weak P2Y-purinergic receptor agonist beta,gamma-methyleneadenosine 5'-triphosphate. These results suggest that BzATP is a full P2Y-purinergic receptor agonist, which after photolysis becomes irreversibly associated with turkey erythrocyte membranes and promotes P2Y-purinergic receptor-mediated guanine nucleotide-dependent activation of phospholipase C.