Identification of Farnesyl Pyrophosphate and N-Arachidonylglycine as Endogenous Ligands for GPR92

Da Young Oh,Jung Min Yoon,Mi Jin Moon,Jong-Ik Hwang,Han Choe,Ju Yeon Lee,Jae Il Kim,Sunoh Kim,Hyewhon Rhim,David K O'Dell,J Michael Walker,Heung Sik Na,Min Goo Lee,Hyuk Bang Kwon,Kyungjin Kim,Jae Young Seong

doi:10.1074/jbc.m708908200

Abstract

A series of small compounds acting at the orphan G protein-coupled receptor GPR92 were screened using a signaling pathway-specific reporter assay system. Lipid-derived molecules including farnesyl pyrophosphate (FPP), N-arachidonylglycine (NAG), and lysophosphatidic acid were found to activate GPR92. FPP and lysophosphatidic acid were able to activate both G(q/11)- and G(s)-mediated signaling pathways, whereas NAG activated only the G(q/11)-mediated signaling pathway. Computer-simulated modeling combined with site-directed mutagenesis of GPR92 indicated that Thr(97), Gly(98), Phe(101), and Arg(267) of GPR92 are responsible for the interaction of GPR92 with FPP and NAG. Reverse transcription-PCR analysis revealed that GPR92 mRNA is highly expressed in the dorsal root ganglia (DRG) but faint in other brain regions. Peripheral tissues including, spleen, stomach, small intestine, and kidney also expressed GPR92 mRNA. Immunohistochemical analysis revealed that GPR92 is largely co-localized with TRPV1, a nonspecific cation channel that responds to noxious heat, in mouse and human DRG. FPP and NAG increased intracellular Ca(2+) levels in cultured DRG neurons. These results suggest that FPP and NAG play a role in the sensory nervous system through activation of GPR92.

Highlights

A series of small compounds acting at the orphan G proteincoupled receptor GPR92 were screened using a signaling pathway-specific reporter assay system
farnesyl pyrophosphate (FPP) and NAG increased intracellular Ca2؉ levels in cultured dorsal root ganglia (DRG) neurons. These results suggest that FPP and NAG play a role in the sensory nervous system through activation of GPR92
The diameter of Ligand Screening for GPR92—We previously established a widely applicable screening system to identify ligands for each DRG neuron was defined as the average of the distance orphan G protein-coupled receptors (GPCRs) [4]

Summary

EXPERIMENTAL PROCEDURES

Chemicals—The chemicals 12-O-tetradecanoylphorbol-13acetate and FPP were purchased from Sigma. Cells were coinfected with adenoviruses containing human GPR92 and an SRE-luc reporter with 50 multiplicity of infection (m.o.i.) under serum-free conditions for 3 h. CV-1 cells were seeded into a 12-well plate and infected with adenoviruses containing human GPR92 with 100 m.o.i. After infection, cells were incubated in M199 medium (Invitrogen) in the presence of 1 ␮Ci/ml myo-[3H]inositol (Amersham Biosciences)/well for 20 h. Cells were washed with 0.5 ml Buffer A (140 mM NaCl, 20 mM HEPES, 4 mM KCl, 8 mM D-glucose, 1 mM MgCl2, 1 mM CaCl2, and 1 mg/ml fatty acid-free bovine serum albumin). Cells were first washed in phosphate-buffered saline and incubated at 37 °C for 20 min in serum-free DMEM containing 1 mM 1-methyl-3-isobutylxanthine. CV-1 cells were infected with 100 m.o.i. adenovirus with human GPR92 and stimulated with different concentrations of FPP, NAG, and LPA in 12-well plates. The Emax values for NAG and LPA are -fold induction at 10 ␮M

FPP NAG LPA

RESULTS

Support for the modeling data

DISCUSSION

While searching for potential