Submitted on August 17, 2004
Accepted on October 20, 2004
Arg333 and Arg334 in the C-terminus of the human P2Y1 receptor are crucial for Gq coupling
Zhongren Ding1, Florin Tuluc2, Kavita R Bandivadekar2, Lili Zhang2, Jianguo Jin2, and Satya P Kunapuli3*
1 Deprtment of Physiology, Temple University School of Medicine, Philadelphia, PA, USA; The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA, USA
2 Deprtment of Physiology, Temple University School of Medicine, Philadelphia, PA, USA
3 Deprtment of Physiology, Temple University School of Medicine, Philadelphia, PA, USA; The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA, USA; Department of Pharmacology, Temple University School of Medicine, Philadelphia, PA, USA
* To whom correspondence should be addressed. E-mail: spk{at}temple.edu.
The P2Y1 ADP receptor activates Gq and causes increases in intracellular calcium through stimulation of phospholipase C. In this study, we investigate the role of the amino acid residues in the carboxyl terminus of the human P2Y1 receptor in Gq activation. Stimulation of CHO-K1 cells stably expressing the wild type human P2Y1 receptor (P2Y1-WT cells), P2Y1-
R340-L373, or P2Y1-D356-L373 with 2-MeSADP caused inositol phosphate production. In contrast, P2Y1-T330-L373, cells expressing a mutant lacking the entire C-terminus, completely lost its response to 2-MeSADP. Similar data were obtained in these cell lines when calcium mobilization was measured upon stimulation with 2-MeSADP, indicating that the 10 amino acids (330TFRRRLSRAT339) in the C-terminus of the human P2Y1 receptor are essential for Gq 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 C-terminus truncation did not drastically affect the conformation of the receptor. CHO-K1 cells expressing a chimeric P2Y12 receptor with the P2Y1 carboxy terminus failed to elicit Gq functional responses, indicating that the P2Y1 carboxy terminus is essential but not sufficient for Gq activation. 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 P2Y1 receptor are essential for Gq coupling.
