Arg333 and Arg334 in the COOH terminus of the human P2Y1 receptor are crucial for Gq coupling.
The P2Y(1) ADP receptor activates G(q) and causes increases in intracellular Ca(2+) concentration through stimulation of PLC. In this study, we investigated the role of the amino acid residues in the COOH terminus of the human P2Y(1) receptor in G(q) activation. Stimulation of Chinese hamster ovary (CHO-K1) cells stably expressing the wild-type human P2Y(1) receptor (P2Y(1)-WT cells), P2Y(1)-DeltaR340-L373, or P2Y(1)-DeltaD356-L373 with 2-methylthio-ADP (2-MeSADP) caused inositol phosphate production. In contrast, cells expressing P2Y(1)-DeltaT330-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 Ca(2+) mobilization upon stimulation with 2-MeSADP, indicating that the 10 amino acids (330TFRRRLSRAT339) in the COOH terminus of the human P2Y(1) receptor are essential for G(q) coupling. Radioligand binding demonstrated that both the P2Y(1)-WT and P2Y(1)-DeltaT330-L373-expressing cells have almost equal binding of [(3)H]MRS2279, a P2Y(1) receptor antagonist, indicating that COOH-terminal truncation did not drastically affect the conformation of the receptor. CHO-K1 cells expressing a chimeric P2Y(12) receptor with the P2Y(1) COOH terminus failed to elicit G(q) functional responses, indicating that the P2Y(1) COOH terminus is essential but not sufficient for G(q) activation. Finally, cells expressing a double-mutant P2Y(1) 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 P2Y(1) receptor are essential for G(q) coupling.
Ding
Zhongren
Z
Department of Physiology, Temple University School of Medicine, 3420 N. Broad St., Philadelphia, PA 19140, USA.
Tuluc
Florin
F
Bandivadekar
Kavita R
KR
Zhang
Lili
L
Jin
Jianguo
J
Kunapuli
Satya P
SP
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D018048:Receptors, Purinergic P2 / Q000235:genetics* / Q000378:metabolism*; D058472:Receptors, Purinergic P2Y1; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013873:Thionucleotides / Q000378:metabolism
C465253:2-chloro-N6-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate; C546382:P2RY1 protein, human; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D011993:Recombinant Fusion Proteins; D013873:Thionucleotides; C002480:methylthio-ADP; D000244:Adenosine Diphosphate; D001120:Arginine; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D000409:Alanine