Identification of a phosphothionate analogue of lysophosphatidic acid (LPA) as a selective agonist of the LPA₃ receptor

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 LPA₁ receptor in yeast cells. OMPT induces intracellular calcium increases efficiently in LPA₃ receptor-expressing Sf9 cells but poorly in LPA₂ receptor-expressing cells. Guanosine 5′-O-(3-[³⁵S]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 LPA₃ receptor. In transiently transfected HEK293 cells, OMPT stimulates mitogen-activated protein kinases through the LPA₃ but not the LPA₁ or LPA₂ receptors. Furthermore, OMPT-induced intracellular calcium mobilization in mammalian cells is efficiently inhibited by the LPA₁/LPA₃ receptor-selective antagonist VPC12249. These results establish that OMPT is an LPA₃-selective agonist. OMPT binding to the LPA₃ 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.