Figure 3
High levels of LPA produce initial agonism followed by functional antagonism of LPA1 after LPA exposure. Receptor internalization was monitored in EGFP-LPA1-B103 neuroblastoma cells combined with fluorescence microscopy and functional calcium imaging. (a, c, e and g) Receptors localized on the cell surface (green signal, dashed arrowheads, a) are rapidly internalized with increasing LPA concentration (green vesicles, arrows, c, e and g). (b and d) After 30 min, cells exposed to BSA and low concentrations of LPA (10 nM) exhibit LPA1 receptor recycling that returns receptors to the cell surface (dashed arrowheads). (f and h) In contrast, exposure to high concentrations of LPA (1 and 10 μM) resulted in prolonged and sustained receptor internalization (arrows), effectively removing receptors from the cell surface. (i–m) To assess calcium signaling, cells were pretreated with BSA or increasing concentrations of LPA for 5 min, washed and after 30 min were challenged with 1 μM LPA followed by assessment of cell signaling viability using ionomycin. Normal levels of LPA-stimulated intracellular Ca2+ (i and j) contrasted with reduced (k) and absent (l) Ca2+ release in cells exposed to higher LPA pretreatment concentrations, indicating functional antagonism of the internalized receptors (m, BSA n=7, 10 nM LPA n=6, 1 μM LPA n=5, 10 μM LPA n=6, mean±s.e.m.). Data were analyzed with Student's two-tailed t-test, **P<0.01, ****P<0.0001. BSA, bovine serum albumin; LPA, lysophosphatidic acid.
