Identification and Characterization of a Novel Lysophosphatidic Acid Receptor, p2y5/LPA6

Keisuke Yanagida,Kayo Masago,Hiroki Nakanishi,Yasuyuki Kihara,Fumie Hamano,Yoko Tajima,Ryo Taguchi,Takao Shimizu,Satoshi Ishii

doi:10.1074/jbc.m808506200

Abstract

p2y5 is an orphan G protein-coupled receptor that is closely related to the fourth lysophosphatidic acid (LPA) receptor, LPA4. Here we report that p2y5 is a novel LPA receptor coupling to the G13-Rho signaling pathway. "LPA receptor-null" RH7777 and B103 cells exogenously expressing p2y5 showed [3H]LPA binding, LPA-induced [35S]guanosine 5'-3-O-(thio)triphosphate binding, Rho-dependent alternation of cellular morphology, and Gs/13 chimeric protein-mediated cAMP accumulation. LPA-induced contraction of human umbilical vein endothelial cells was suppressed by small interfering RNA knockdown of endogenously expressed p2y5. We also found that 2-acyl-LPA had higher activity to p2y5 than 1-acyl-LPA. A recent study has suggested that p2y5 is an LPA receptor essential for human hair growth. We confirmed that p2y5 is a functional LPA receptor and propose to designate this receptor LPA6.

Highlights

Cell Aggregation of B103-p2y5 Cells in Serum-containing Medium—An orphan receptor p2y5 shares the highest amino acid sequence homology with LPA4 among all G protein-coupled receptors (GPCRs) [12], which led us to hypothesize that p2y5 is an additional LPA receptor. We tested this hypothesis by detecting Ca2ϩ mobilization or the changes of intracellular cAMP level using RH7777 cells and B103 cells stably overexpressing p2y5 (RH7777-p2y5 cells and B103-p2y5 cells, respectively) These cell lines were selected because they lack endogenous responses to LPA in these assays [18, 21]
We previously showed that B103 cells expressing LPA4 formed aggregates through Rho in serum-containing medium, which is abundant in LPA [18]
We consistently observed that the 2-oleoyl-LPA prepared by our method showed higher activity than 1-oleoyl-LPA in the Ca2ϩ mobilization of RH7777 cells stably expressing LPA3 but not in the cells expressing LPA1 (Fig. S2). cAMP assays gave consistent results (Fig. 6C, left and middle), we examined the effect of the position of fatty acid chain on p2y5 activation in the cAMP assay with Gs/13-transfected B103 cells after pertussis toxin (PTX) treatment

Summary

Introduction

[35S]GTP␥S Binding to the G␣13 Protein—The membrane fraction (40 ␮g of protein) from RH7777 cells transiently expressing p2y5 was incubated in 100 ␮l of GTP␥S binding buffer containing 0.5 nM [35S]GTP␥S with or without 10 ␮M 1-oleoyl-LPA for 30 min at 30 °C. Flow cytometry analysis proved the high expression levels of p2y5 in these cells (Fig. 1A), we could not observe any p2y5-dependent signaling by 1-oleoyl-LPA (data not shown). With membranes from RH7777 cells transiently expressing p2y5, 10 ␮M 1-oleoyl-LPA induced an ϳ4-fold increase in [35S]GTP␥S binding to G13 proteins, whereas

Results

Conclusion