Figure 2.

The extreme C-terminus of the P2Y₁₂ receptor is required for normal agonist-induced regulation of receptor function. (A-B) P2Y₁₂ desensitization and subsequent resensitization were assessed by comparing agonist (ADP; 10μM)–dependent inhibition of forskolin (1μM; 10 minutes)–stimulated adenylyl cyclase activity before (control) and after pretreatment with either ADP alone (10nM; 15 minutes; desens on graph) or after subsequent removal of desensitizing ADP with apyrase (0.2 U/mL; 15 minutes; resens on graph). Data represent means ± SEMs of 6 independent experiments. (A) Raw data are expressed as pmol cAMP/mg protein. (B) Data are expressed as percentage of initial P2Y₁₂ response. *Statistical significance at P < .05 for data compared with respective densitized wild-type control (Mann-Whitney U test). #Statistical significance at P < .05 for resensitized compared with respective resensitized control (Mann-Whitney U test). (C-D) Phosphorylation of full-length and mutant HA-P2Y₁₂ was assessed in CHO cells stably expressing receptor constructs. Cells were treated with ADP (10μM; 5 minutes) or vehicle alone, and HA-tagged receptors were immunoprecipitated from membrane lysates and run on SDS-PAGE before transfer to nitrocellulose membranes. Specific phosphorylated bands between 50 and 75 kDa not present in vector-alone pcNEO-transfected controls were subsequently identified with a phosphothreonine-specific Ab. Similar amounts of receptor immunoprecipitation were confirmed by reprobing membranes with a polyclonal anti-HA Ab/HRP-conjugated anti–rabbit IgG and visualization by ECL. Receptor immunoprecipitation was also confirmed by reprobing with a P2Y₁₂-specific N-terminal Ab. Data are representative of 3 individual experiments. (C) Densitometric analysis of 3 experiments was performed, and data were expressed as fold increase over basal phosphorylation