The orphan GPCR GPR87 was deorphanized and shown to be a lysophosphatidic acid receptor

https://doi.org/10.1016/j.bbrc.2007.09.063Get rights and content

Abstract

In CHO cells stably expressing the GPR87 fused with a G16α protein, lysophosphatidic acid (LPA) evoked an intracellular Ca2+ increase in a high affinity manner. The Ca2+ increase was reversibly blocked by the LPA receptor antagonists and inhibited by pretreatment of the cells with GPR87-specific siRNAs. GPR87 was shown to be closer to the P2Y and P2Y-related receptors than LPA receptors by ClustalW analyses. However, none of nucleotides and their derivatives activated GPR87. The human gpr87 is located on the chromosome 3q25 in a cluster containing p2y12,13,14. RT-PCR analysis showed that the mouse GPR87 was expressed in placenta, ovary, testis, prostate, brain, and skeletal muscle. The 3D model of GPR87–LPA complex indicated that the ligand interacted with R115 and K296 of GPR87, which are well conserved in the P2Y receptors. These results suggest that the GPR87 is a LPA receptor which evolved from a common ancestor of P2Y receptors.

Section snippets

Materials and methods

The cDNA coding human GPR87 was from GFC-Array™ (Origene, Rockville) and the human G16α in pCIS plasmid was kindly provided by Prof. Haga T. in Gakushuin University. The human GPR87-G16α fusion gene was generated by a two-step PCR protocol using KOD DNA polymerase (TOYOBO, Osaka). The gene was cloned into the pcDNA5/FRT (Invitrogen, Carlsbad), and transfected into Flp-InTM CHO cells (Invitrogen) using Lipofectamine™ 2000 (Invitrogen). Cell populations stably expressing the fusion genes were

Results

From the extract of the CHO cells transformed with the pcDNA5 vector containing GPR87-G16α fusion gene, we detected a clear band of GPR87-G16α fusion gene by RT-PCR analysis. This analysis demonstrated that the transfected cells effectively expressed the gene to the point that the level of the GPR87 was as high as the major cytoskeleton protein β-actin. Furthermore, a 84 kDa protein-band corresponding to the fusion protein was identified by Western blot analysis (suppl).

Using the recombinant

Discussion

In our previous report, ADP, PRPP, and CysLTE4 were selected as a P2Y12 ligand by in silico screening, and subsequent in vitro experiments showed that they interacted with the P2Y12 receptor in high affinity manners. In the report, we showed GPR87 was classified in the P2Y12 subgroup that contains P2Y12, P2Y13, P2Y14, CysLT1, and CysLT2 receptors [5]. Joost and Methner have proposed that GPR87 is closely related to the P2Y14 receptor to which UDP-glucose specifically binds [18]. Thus we

Acknowledgments

This work was supported by the Ministry of Education, Culture, Sports, Sciences and Technology of Japan. We thank to Associate Prof. E. Cooper of Ritsumeikan University for help in preparing this manuscript.

References (26)

  • T. Suzuki et al.

    Regulation of pharmacology by hetero-oligomerization between A1 adenosine receptor and P2Y2 receptor

    Biochem. Biophys. Res. Commun.

    (2006)
  • D. Meyer zu Heringdorf et al.

    Lysophospholipid receptors: signaling, pharmacology and regulation by lysophospholipid metabolism

    Biochim. Biophys. Acta

    (2007)
  • I. von Kugelgen

    Pharmacological profiles of cloned mammalian P2Y-receptor subtypes

    Pharmacol. Ther.

    (2006)

    • Cited by (0)

    View full text
    Copyright © 2007 Elsevier Inc. All rights reserved.