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RECEPTORS AND SIGNAL TRANSDUCTION

Arg333 and Arg334 in the COOH terminus of the human P2Y₁ receptor are crucial for G_q coupling

¹Department of Physiology, ²Department of Pharmacology, and ³The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania

Submitted 16 August 2004 ; accepted in final form 20 October 2004

The P2Y₁ ADP receptor activates G_q and 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 P2Y₁ receptor in G_q activation. Stimulation of Chinese hamster ovary (CHO-K1) cells stably expressing the wild-type human P2Y₁ receptor (P2Y₁-WT cells), P2Y₁-R340-L373, or P2Y₁-D356-L373 with 2-methylthio-ADP (2-MeSADP) caused inositol phosphate production. In contrast, cells expressing P2Y₁-T330-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 P2Y₁ receptor are essential for G_q coupling. Radioligand binding demonstrated that both the P2Y₁-WT and P2Y₁-T330-L373-expressing cells have almost equal binding of [³H]MRS2279, a P2Y₁ receptor antagonist, indicating that COOH-terminal truncation did not drastically affect the conformation of the receptor. CHO-K1 cells expressing a chimeric P2Y₁₂ receptor with the P2Y₁ COOH terminus failed to elicit G_q functional responses, indicating that the P2Y₁ COOH terminus is essential but not sufficient for G_q activation. Finally, cells expressing a double-mutant P2Y₁ receptor (R333A/R334A) in the conserved BBXXB region of the COOH terminus of the G_q-activating P2Y receptors completely lost their functional ability to activate G_q. We conclude that the two arginine residues (R333R334) in the COOH terminus of the human P2Y₁ receptor are essential for G_q coupling.

carboxyl terminus; adenosine diphosphate; truncation; inositol phosphate

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