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. 2001 Sep;134(1):6-9.
doi: 10.1038/sj.bjp.0704278.

Assessment of agonism at G-protein coupled receptors by phosphatidic acid and lysophosphatidic acid in human embryonic kidney 293 cells

F Alderton  1 , B Sambi, R Tate, N J Pyne, S Pyne
Affiliations

Assessment of agonism at G-protein coupled receptors by phosphatidic acid and lysophosphatidic acid in human embryonic kidney 293 cells

F Alderton et al. Br J Pharmacol. 2001 Sep.

Abstract

Several different molecular species of phosphatidic acid (PA) bind to a G-protein coupled receptor (GPCR) to induce activation of the p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) pathway in HEK 293 cells. PA is active at low nanomolar concentrations and the response is sensitive to pertussis toxin (which uncouples GPCRs from G(i/o)). The de-acylated product of PA, lysophosphatidic acid (LPA), which binds to members of the endothelial differentiation gene (EDG) family of receptors also stimulated p42/p44 MAPK in a pertussis toxin sensitive manner, but with an approximately 100 - 1000 fold lower potency compared with the different molecular species of PA. RT - PCR using gene-specific primers showed that HEK 293 cells express EDG2 and PSP24, the latter being a lipid binding GPCR out with the EDG cluster. We conclude that PA is a novel high potency GPCR agonist.

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Figures

Figure 1
Figure 1
PA regulation of the p42/p44 MAPK pathway. HEK 293 cells were pre-treated with and without pertussis toxin (0.1 μg ml−1, 18 h) or suramin (50 μM, 10 min), prior to stimulation with PA (0.3 – 30 nM, 10 min) or PMA (1 μM, 10 min). Western blots probed with anti-phospho p42/p44 MAPK and anti-p42 MAPK antibodies showing (a) the effect of pertussis toxin on the stimulation of p42/p44 MAPK by dioctanoyl PA (3 nM) and PMA; (b) the concentration-dependent effect of dioctanoyl PA (1), dioleoyl PA (2), dipalmitoyl PA (3) and stearoyl/arachidonoyl PA (4); (c) the effect of suramin on the stimulation of p42/p44 MAPK by dioctanoyl PA (3 nM), dipalmitoyl PA (30 nM), stearoyl-arachidonoyl PA (30 nM), dioleoyl PA (30 nM) and PMA. Total p42 MAPK immunostaining showed equal loading of protein in each sample (data not shown). These are representative results of three independent experiments.
Figure 2
Figure 2
LPA regulation of the p42/p44 MAPK pathway. HEK 293 cells were pre-treated with and without pertussis toxin (0.1 μg ml−1, 18 h) prior to stimulation with LPA (1 – 100 μM, 10 min). Western blots probed with anti-phospho p42/p44 MAPK antibodies showing (a) the concentration-dependent effect of LPA; (b) the effect of pertussis toxin on the LPA- (5 μM) dependent stimulation of p42/p44 MAPK. These are representative results of three independent experiments.
Figure 3
Figure 3
RT – PCR of EDG2, EDG4, EDG7 and PSP24. (a) RT – PCR of EDG2 but not EDG4 or EDG7 transcripts. (b) RT – PCR of PSP24 transcripts.
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