Phosphorylation and Internalization of Lysophosphatidic Acid Receptors LPA1, LPA2, and LPA3.

The lysophosphatidic acid receptors LPA1, LPA2, and LPA3 were individually expressed in C9 cells and their signaling and regulation were studied. Agonist-activation increases intracellular calcium concentration in a concentration-dependent fashion. Phorbol myristate acetate markedly inhibited LPA1- and LPA3-mediated effect, whereas that mediated by LPA2 was only partially diminished; the actions of the phorbol ester were inhibited by bisindolylmaleimide I and by overnight incubation with the protein kinase C activator, which leads to down regulation of this protein kinase. Homologous desensitization was also observed for the three LPA receptors studied, with that of LPA2 receptors being consistently of lesser magnitude; neither inhibition nor down-regulation of protein kinase C exerted any effect on homologous desensitization. Activation of LPA1-3 receptors induced ERK 1/2 phosphorylation; this effect was markedly attenuated by inhibition of epidermal growth factor receptor tyrosine kinase activity, suggesting growth factor receptor transactivation in this effect. Lysophosphatidic acid and phorbol myristate acetate were able to induce LPA1-3 phosphorylation, in time- and concentration-dependent fashions. It was also clearly observed that agonists and protein kinase C activation induced internalization of these receptors. Phosphorylation of the LPA2 subtype required larger concentrations of these agents and its internalization was less intense than that of the other subtypes.Our data show that these three LPA receptors are phosphoproteins whose phosphorylation state is modulated by agonist-stimulation and protein kinase C-activation and that differences in regulation and cellular localization exist, among the subtypes

Effect of LPA on intracellular calcium concentration ([Ca²⁺]_i).

<p>Wild type C9 cells (panel A) or overexpressing LPA₁ (panel B), LPA₂ (panel C) or LPA₃ (panel D) were stimulated by different concentrations of LPA. Plotted are the increases in intracellular calcium as mean ± S. E. M. of 4–5 experiments using different cell preparations.</p

Time-courses of the effects of LPA and PMA on LPA_1–3 receptor phosphorylation.

<p>Cells overexpressing LPA₁ (black, circles), LPA₂ (blue, squares) or LPA₃ (red, triangles) receptors were incubated for the times indicated in the presence of 1 μM LPA (Panel A) or 1 μM PMA (Panel B). Plotted are the percentage of baseline phosphorylations as mean ± S. E. M. of 4–5 experiments using different cell preparations. Representative autoradiographs are presented for the different receptor subtypes.</p

Effects of LPA and PMA on LPA_1–3 receptor internalization (60 and 120 min).

<p>Cells overexpressing LPA₁ (panel A), LPA₂ (panel B) or LPA₃ (Panel C) receptors were incubated in the absence of any agent (Baseline), for 10 min in the presence of 1 μM LPA, or for 2 min in the presence of 1 μM PMA. After this incubation cells were extensively washed and further incubated for the times indicated (60 or 120 min) Plotted is membrane-associated fluorescence (arbitrary units) as the mean ± S. E. M. of 5 different fields of 3 experiments using different cell preparations. * p <0.001 vs. baseline (B), ** p < 0.01 vs. baseline (B), *** p < 0.05 vs. baseline (B).</p

Effect of PKC down regulation on heterologous (PMA-induced) or homologous (LPA-induced) desensitization.

<p>Cells overexpressing LPA_1–3 receptors were preincubated overnight with 1 μM PMA, and then subjected to the desensitization protocols (indicated under the Experimental section and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140583#pone.0140583.g002" target="_blank">Fig 2</a>) using 1 μM PMA (2 min) or 1 μM LPA (10 min). Cells were challenged with 1 μM LPA, and the increase in intracellular free calcium concentration was determined. Plotted are the increases in calcium as the percentage of that obtained in cells preincubated without any agent as mean ± S. E. M. of 5–7 experiments using different cell preparations. *p < 0.001 vs baseline; **p <0.05 vs baseline.</p

Images of the effects of LPA and PMA on LPA_1–3 receptor internalization.

<p>Fluorescent confocal images of cells overexpressing LPA₁ (column A), LPA₂ (column B) or LPA₃ (column C) receptors were incubated in the absence of any agent (Baseline) or for 30 or 60 min in the presence of 1 μM LPA or 1 μM PMA. Images are representative of data of 3–4 experiments using different cell preparations. Bars 15 μm.</p

Images of the effects of LPA and PMA on LPA_1–3 receptor internalization (60 and 120 min).

<p>Fluorescent confocal images of cells overexpressing LPA₁ (column A), LPA₂ (column B) or LPA₃ (column C) receptors. Cells were incubated in the absence of any agent (Baseline), for 10 min in the presence of 1 μM LPA, or for 2 min in the presence of 1 μM PMA. After this incubation cells were extensively washed and further incubated for the times indicated. Images are representative of data of 3 experiments using different cell preparations. Bars 10 μm.</p

Effects of LPA and PMA on LPA_1–3 receptor internalization.

<p>Cells overexpressing LPA₁ (panel A), LPA₂ (panel B) or LPA₃ (Panel C) receptors were incubated for 30 or 60 min in the presence of 1 μM LPA or 1 μM PMA. Plotted is membrane-associated fluorescence (arbitrary units) as the mean ± S. E. M. of 5 different fields of 3–4 experiments using different cell preparations. * p <0.001 vs. baseline (B), ** p < 0.01 vs. baseline (B), *** p < 0.05 vs. baseline (B).</p

Transactivation of EGF receptors in LPA-induced ERK 1/2 phosphorylation.

<p>Cells overexpressing LPA₁ (panels A and D), LPA₂ (panels B and E) or LPA₃ (panels C and F) receptors were incubated in the absence or presence of 10 μM AG1478 (AG) for 30 min and then challenged with 1 μM LPA (panels A-C) or 100 ng/ml EGF (panels D-F) for 5 min; incubation was terminated and phospho-ERK 1/2 (pERK) and total ERK 1/2 (ERK) were assayed by Western blotting. Plotted are the increases in phospho-ERK 1/2 as mean ± S. E. M. of 4–5 experiments using different cell preparations. Representative Western blots are presented for the different receptor subtypes. *p < 0.001 vs. baseline (B); ** p <0.001 vs. LPA or EGF alone.</p

Effect of PKC inhibition on heterologous (PMA-induced) or homologous (LPA-induced) desensitization.

<p>Cells overexpressing LPA_1–3 receptors were preincubated for 15 min in the presence of the PKC inhibitor, bisindolylmaleimide I (BIM), and then subjected to the desensitization protocols (indicated under the Experimental section and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140583#pone.0140583.g002" target="_blank">Fig 2</a>), using 1 μM PMA or 1 μM LPA. Cells were challenged with 1 μM LPA and the increase in intracellular free calcium concentration was determined. Plotted are the increases in calcium as the percentage of that obtained in cells preincubated without any agent as mean ± S. E. M. of 6–8 experiments using different cell preparations. *p < 0.001 vs. baseline.</p