Identification of a Phosphothionate Analogue of Lysophosphatidic Acid (LPA) as a Selective Agonist of the LPA3 Receptor

Yutaka Hasegawa,James R Erickson,Graham J Goddard,Shuangxing Yu,Shuying Liu,Kwai Wa Cheng,Astrid Eder,Koji Bandoh,Junken Aoki,Renata Jarosz,Andrew D Schrier,Kevin R Lynch,Gordon B Mills,Xianjun Fang

doi:10.1074/jbc.m209168200

Abstract

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid mediator that acts through G protein-coupled receptors. Most cell lines in culture express one or more LPA receptors, making it difficult to assign a response to specific LPA receptors. Dissection of the signaling properties of LPA has been hampered by lack of LPA receptor subtype-specific agonists and antagonists. The present study characterizes an ester-linked thiophosphate derivative (1-oleoyl-2-O-methyl-rac-glycerophosphothionate, OMPT) of LPA. OMPT is a functional LPA analogue with potent mitogenic activity in fibroblasts. In contrast to LPA, OMPT does not couple to the pheromone response through the LPA(1) receptor in yeast cells. OMPT induces intracellular calcium increases efficiently in LPA(3) receptor-expressing Sf9 cells but poorly in LPA(2) receptor-expressing cells. Guanosine 5'-O-(3-[(35)S]thio)triphosphate binding assays in mammalian cells showed that LPA exhibits agonistic activity on all three LPA receptor subtypes, whereas OMPT has a potent agonistic effect only on the LPA(3) receptor. In transiently transfected HEK293 cells, OMPT stimulates mitogen-activated protein kinases through the LPA(3) but not the LPA(1) or LPA(2) receptors. Furthermore, OMPT-induced intracellular calcium mobilization in mammalian cells is efficiently inhibited by the LPA(1)/LPA(3) receptor-selective antagonist VPC12249. These results establish that OMPT is an LPA(3)-selective agonist. OMPT binding to the LPA(3) receptor in mammalian cells is sufficient to elicit multiple responses, including activation of G proteins, calcium mobilization, and activation of mitogen-activated protein kinases. Thus OMPT offers a powerful probe for the dissection of LPA signaling events in complex mammalian systems.

Highlights

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid mediator that acts through G protein-coupled receptors
By virtue of these approaches combined with assays in mammalian cells such as guanosine 5Ј-O-(3-[35S]thio)triphosphate (GTP[␥-35S]) binding assay [31], we have demonstrated that OMPT, at low concentrations (Յ100 nM), triggers cellular responses through the LPA3 but not the LPA1 or LPA2 receptors
To determine whether OMPT exhibits agonistic activities, we first assessed the ability of OMPT to increase intracellular calcium change in mammalian cells, a standard assay for LPA activity

Summary

EXPERIMENTAL PROCEDURES

Reagents—LPA (18:1) and S1P were obtained from Avanti Polar Lipids (Alabaster, AL) and Calbiochem, respectively. Cytoplasmic [Ca2ϩ]i Assay—After starvation in serum-free medium for 12–24 h, C3H10T1/2 cells were harvested and loaded with 1 ␮M Indo-1 AM in phosphate-buffered saline (PBS) for 30 min at 37 °C. After cells were loaded with 1 ␮M Indo-1 AM in PBS for 30 min at 27 °C, cytoplasmic [Ca2ϩ]i was determined at 27 °C in the same [Ca2ϩ]i assay buffer as described above for C3H10T1/2 cells. Membranes containing 5 ␮g of protein were incubated in 0.1 ml of GTP-binding buffer (50 mM Hepes, 100 mM NaCl, 10 mM MgCl2, pH 7.5) containing 5 ␮g of saponin, 0.1% fatty acid-free BSA, 10 ␮M GDP, 0.1 nM GTP[␥-35S] (1200 Ci/mmol), and indicated concentrations of LPA or OMPT for 30 min at 30 °C. HA-Erk as described previously [33]

RESULTS

After infection with recombinant baculovirus expressing

DISCUSSION