Guanine nucleotide-sensitive interaction of a radiolabeled agonist with a phospholipase C-linked P(2y)-purinergic receptor

Analogs of ATP and ADP produce a guanine nucleotide-dependent activation of phospholipase C in turkey erythrocyte membranes with pharmacological properties consistent with those of a P(2y)-purinergic receptor (Boyer, J.L., Downes, C.P., and Harden, T.K. (1989) J. Biol. Chem. 264, 884-890). This study describes the interaction of adenosine-5'-O-2-thio[³⁵S] diphosphate ([³⁵S]ADPβS) with this putative (P(2y)-purinergic receptor on purified plasma membranes prepared from turkey erythrocytes. In binding assays performed at 30°C, the association rate constant of [³⁵S]ADPβS was 1.1 x 10⁷ M^-1 min^-1 and the dissociation rate constant was 3.8 x 10^-1 min^-1. [³⁵S]ADPβS bound with high affinity (K(d) = 6-10 nM) to an apparently homogeneous population of sites (B(max) = 2-4 pmol/mg protein). ATP and ADP analogs (2-methylthio ATP, ADPβS, ATP, ADP, 5'-adenylyl imidodiphosphate, α,β-omethylene adenosine-5'-triphosphate, and β,γ-methylene adenosine 5'-triphosphate) inhibited the binding of [³⁵S]ADPβS with properties consistent with ligand interaction by simple law of mass action kinetics at a single site. The rank order of potency for inhibition of [³⁵S]ADPβS binding was identical to the potency order observed for these same agonists for stimulation of phospholipase C in turkey erythrocyte ghosts. Guanine nucleotides inhibited [³⁵S]ADPβS binding in a noncompetitive manner with the following potency order: guanosine 5'-O-(3-thiotriphosphate) > 5'-guanylyl imidodiphosphate > GTP = GDP > guanosine 5'-O-2-(thiodiphosphate). The data are consistent with the idea that [³⁵S]ADPβS may be used to radiolabel the P(2y)-purinergic receptor linked to activation of phospholipase C in turkey erythrocyte membranes. In addition, interaction of radiolabeled agonist with the receptor is modified by guanine nucleotides, providing evidence that an agonist-induced receptor/guanine nucleotide regulatory protein complex may be involved in P(2y)-receptor action.