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

Abstract

Several di erent 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 di erentiation gene (EDG) family of receptors also stimulated p42/p44 MAPK in a pertussis toxin sensitive manner, but with an *100 ± 1000 fold lower potency compared with the di erent molecular species of PA. RT ± PCR using gene-speci®c 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.

Ceramide Synthesis Is Modulated by the Sphingosine Analog FTY720 via a Mixture of Uncompetitive and Noncompetitive Inhibition in an Acyl-CoA Chain Length-de pend ent Manner

“…Finally, to determine that the changes in ceramide synthesis in cultured cells were not due to the conversion of FTY720 to FTY720-P (9), which could bind to an S1PR at the cell surface and thus influence ceramide synthesis, we examined the effect of FTY720 in the presence of suramin, an inhibitor of G-protein-coupled receptors (31). No changes were observed in ceramide synthesis after incubation with FTY720 in the absence or presence of suramin (Fig.…”

Phosphorylation of P-Rex1 by the Cyclic AMP-dependent Protein Kinase Inhibits the Phosphatidylinositiol (3,4,5)-Trisphosphate and Gβγ-mediated Regulation of Its Activity

“…Accordingly, HEK293T cells were transfected with an expression plasmid encoding for an EE-tagged P-Rex1 and stimulated via their endogenous LPA and ␤-adrenergic receptors, which can couple to G i and G s , respectively (35)(36)(37). The data presented in Fig.…”

Novel clusters of receptors for sphingosine‐1‐phosphate, sphingosylphosphorylcholine, and (lyso)‐phosphatidic acid: New receptors for “Old” ligands

“…It has, for example, been reported to be involved in membrane recruitment of Raf-1, which contains a specific PA binding region [Rizzo et al, 2000]. Besides its action as intracellular second messenger studies suggested that PA may signal via cell surface GPCRs either in the micromolar [Sliva et al, 2000] or nanomolar concentration range [Alderton et al, 2001]. Alderton et al [2001] have shown that PA and related molecular species such as doPA activate, via a Ga i/o -dependent mechanism, the p42/44 mitogen-activated protein kinase (MAPK) pathway in HEK293 cells.…”

“…Besides its action as intracellular second messenger studies suggested that PA may signal via cell surface GPCRs either in the micromolar [Sliva et al, 2000] or nanomolar concentration range [Alderton et al, 2001]. Alderton et al [2001] have shown that PA and related molecular species such as doPA activate, via a Ga i/o -dependent mechanism, the p42/44 mitogen-activated protein kinase (MAPK) pathway in HEK293 cells. They suggested GPR45 to be a possible candidate but did not test PA for functional activity on GPR45 in this report.…”

“…Future studies will show whether OGR1 and GPR4 turn out to be proton sensing receptors rather than high affinity SPC receptors and those observations may determine the need to hunt for additional SPC receptors. PA has been reported to signal through an endogeneous receptor in HEK293 cells in the nanomolar concentration range [Alderton et al, 2001]. However, only one cell surface PA receptor is known so far requiring micromolar PA concentrations for activation [Niedernberg et al, 2003a].…”