LPA3 receptors were expressed in TREx HEK 293 cells, and their signaling and phosphorylation were studied. The agonist, lysophosphatidic acid (LPA), increased intracellular calcium and ERK phosphorylation through pertussis toxin-insensitive processes. Phorbol myristate acetate, but not LPA, desensitizes LPA3-mediated calcium signaling. The agonists and the phorbol ester-induced LPA3 internalization. Pitstop 2 (clathrin heavy chain inhibitor) markedly reduced LPA-induced receptor internalization; in contrast, phorbol ester-induced internalization was only delayed. LPA induced rapid β-arrestin-LPA3 receptor association. The agonist and the phorbol ester induced marked LPA3 receptor phosphorylation, and phosphorylation sites were detected using mass spectrometry. Phosphorylated residues were detected in the intracellular loop 3 (S221, T224, S225, and S229) and in the carboxyl terminus (S321, S325, S331, T333, S335, Y337, and S343). Interestingly, phosphorylation sites are within sequences predicted to constitute β-arrestin binding sites. These data provide insight into LPA3 receptor signaling and regulation.

Lysophosphatidic acid (LPA) type 3 (LPA3) receptor mutants were generated in which the sites detected phosphorylated were substituted by non-phosphorylatable amino acids. Substitutions were made in the intracellular loop 3 (IL3 mutant), the carboxyl terminus (Ctail), and both domains (IL3/Ctail). The wild-type (WT) receptor and the mutants were expressed in T-REx HEK293 cells, and the consequences of the substitutions were analyzed employing different functional parameters. Agonist- and LPA-mediated receptor phosphorylation was diminished in the IL3 and Ctail mutants and essentially abolished in the IL3/Ctail mutant, indicating that the main sites were substituted and distributed in both domains. The WT and mutant receptors increased intracellular calcium and ERK 1/2 phosphorylation in response to LPA and PMA. An increase in baseline ERK phosphorylation was detected in the IL3/Ctail mutant. LPA and PMA-induced receptor interaction with β-arrestin 2 and LPA3 internalization were severely diminished in the mutants. Mutant-expressing cells also exhibit increased baseline proliferation and response to different stimuli, which were inhibited by the antagonist Ki16425, suggesting a role of LPA receptors in this process. Migration in response to different attractants was markedly increased in the Ctail mutant, which the Ki16425 antagonist also attenuated.