Arg333 and Arg334 in the C-Terminus of the Human Platelet P2Y₁ Receptor Are Crucial for Gq Coupling.

ADP-induced platelet aggregation plays an important role in hemostasis and thrombosis. Human platelets express two ADP receptors: the P2Y₁ and P2Y₁₂ receptors. The Gq-activating P2Y₁ receptor plays an important role in ADP-induced platelet shape change, aggregation, and thromboxane A2 generation. In this study, we investigated the role of the carboxyl terminus of the human P2Y₁ receptor in Gq activation. Human P2Y₁ receptors, either wild type (P2Y₁-WT) or a mutant in which the C-terminus was truncated (P2Y₁-ΔT330-L373), were stably expressed with an HA-tag at the N-terminus in CHO-K1 cells. Stimulation of P2Y₁-WT cells with 2-MeSADP caused inositol phosphate production and mobilization of calcium from intracellular stores. In contrast, P2Y₁-ΔT330-L373 completely lost its response to 2-MeSADP, indicating that the C-terminus of the human P2Y₁ receptor is essential for the activation of Gq. CHO-K1 cells expressing a chimeric P2Y₁₂ receptor with the P2Y₁ carboxyl terminus failed to elicit Gq functional responses, indicating that the P2Y₁ carboxyl terminus is essential but not sufficient for Gq activation. Radioligand binding demonstrated that both the P2Y₁-WT- and P2Y₁-ΔT330-L373- expressing cells have almost equal binding of [3H]MRS2279, a P2Y₁ receptor antagonist, indicating that C-terminus truncation did not drastically affect the conformation of the receptor. Two additional truncation mutants in the C-terminus, P2Y₁-ΔR340-L373 and P2Y₁-ΔD356-L373, were expressed in CHO-K1 cells, and responded to 2-MeSADP with functional responses. These results indicate that the 10 amino acids (330TFRRRLSRAT339) in the C-terminus of the human P2Y₁ receptor are essential for Gq coupling. Finally, the cells expressing a double mutant P2Y₁ receptor (R333A and R334A), in the conserved BBXXB region in the C-terminus of the Gq-activating P2Y receptors, completely lost its functional ability to activate Gq. We conclude that the two Arg residues (R333R334) in the C-terminus of the human platelet P2Y₁ receptor are essential for Gq coupling and subsequent platelet activation.