Arg333 and Arg334 in the COOH terminus of the human P2Y1receptor are crucial for Gqcoupling

Abstract

The P2Y1ADP receptor activates Gqand causes increases in intracellular Ca2+concentration through stimulation of PLC. In this study, we investigated the role of the amino acid residues in the COOH terminus of the human P2Y1receptor in Gqactivation. Stimulation of Chinese hamster ovary (CHO-K1) cells stably expressing the wild-type human P2Y1receptor (P2Y1-WT cells), P2Y1-ΔR340-L373, or P2Y1-ΔD356-L373 with 2-methylthio-ADP (2-MeSADP) caused inositol phosphate production. In contrast, cells expressing P2Y1-ΔT330-L373, a mutant lacking the entire COOH terminus, completely lost their response to 2-MeSADP. Similar data were obtained by using these cell lines and measuring Ca2+mobilization upon stimulation with 2-MeSADP, indicating that the 10 amino acids (330TFRRRLSRAT339) in the COOH terminus of the human P2Y1receptor are essential for Gqcoupling. Radioligand binding demonstrated that both the P2Y1-WT and P2Y1-ΔT330-L373-expressing cells have almost equal binding of [3H]MRS2279, a P2Y1receptor antagonist, indicating that COOH-terminal truncation did not drastically affect the conformation of the receptor. CHO-K1 cells expressing a chimeric P2Y12receptor with the P2Y1COOH terminus failed to elicit Gqfunctional responses, indicating that the P2Y1COOH terminus is essential but not sufficient for Gqactivation. Finally, cells expressing a double-mutant P2Y1receptor (R333A/R334A) in the conserved BBXXB region of the COOH terminus of the Gq-activating P2Y receptors completely lost their functional ability to activate Gq. We conclude that the two arginine residues (R333R334) in the COOH terminus of the human P2Y1receptor are essential for Gqcoupling.