Guanine nucleotide-sensitive interaction of a radiolabeled agonist with a phospholipase C-linked P2y-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 P2y-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]ADP beta S) with this putative P2y-purinergic receptor on purified plasma membranes prepared from turkey erythrocytes. In binding assays performed at 30 degrees C, the association rate constant of [35S] was 1.1 x 10(7) M-1 min-1 and the dissociation rate constant was 3.8 x 10(-2) min-1. [35S]ADP beta S bound with high affinity (Kd = 6-10 nM) to an apparently homogeneous population of sites (Bmax = 2-4 pmol/mg protein). ATP and ADP analogs (2-methylthio ATP, ADP beta S, ATP, ADP, 5'-adenylyl imidodiphosphate, alpha, beta-methylene adenosine-5'-triphosphate, and beta, gamma-methylene adenosine 5'-triphosphate) inhibited the binding of [35S]ADP beta S 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]ADP beta S 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]ADP beta S binding in a noncompetitive manner with the following potency order: guanosine 5'-O-(3-thiotriphosphate) greater than 5'-guanylyl imidodiphosphate greater than GTP = GDP greater than guanosine 5'-O-2-(thiodiphosphate). The data are consistent with the idea that [35S]ADP beta S may be used to radiolabel the P2y-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 P2y-receptor action.
Cooper
C L
CL
Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill 27599.
Morris
A J
AJ
Harden
T K
TK
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D004789:Enzyme Activation; D004910:Erythrocyte Membrane / Q000502:physiology; D019204:GTP-Binding Proteins / Q000502:physiology*; D006150:Guanine Nucleotides / Q000494:pharmacology; D007700:Kinetics; D011983:Receptors, Purinergic / Q000502:physiology*; D013873:Thionucleotides / Q000378:metabolism*; D014422:Turkeys; D010738:Type C Phospholipases / Q000502:physiology*
D006150:Guanine Nucleotides; D011983:Receptors, Purinergic; D013873:Thionucleotides; C030812:adenosine 5'-O-(2-thiodiphosphate); D000244:Adenosine Diphosphate; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins