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

Alcántara-Hernández R, Hernández-Méndez A, Campos-Martínez GA, Meizoso-Huesca A, García-Sáinz JA - PLoS ONE (2015)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Apartado Postal 70-248, México D.F., México 04510.

ABSTRACT

Results: 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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus

Effect of preincubation with PMA (heterologous desensitization) or LPA (homologous desensitization) on LPA-induced intracellular calcium concentration ([Ca2+]i) using high agonist concentrations.Cells overexpressing LPA1 (panel A, black), LPA2 (panel B, blue) or LPA3 (panel C, red) receptors were preincubated in the absence or presence of 1 μM LPA for 10 min, extensively washed, then challenged with the indicated concentrations of 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 and challenged with 1 μM LPA (C 1 in the abscisa) (% of control) as mean ± S. E. M. of 6 experiments using different cell preparations. In the right panels concentration response curves are plotted. Data from Fig 1 (without pre-stimulation) were normalized and re-plotted as percentage of the maximal response (solid symbols and continuous connecting lines) together with those in the left panels of this figure, normalized in the same way (open symbols, dotted connected lines). The response of cells preincubated with 1 μM PMA for 2 min, washed, and then challenged with to 100 μM LPA is also presented (solid triangles).
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pone.0140583.g003: Effect of preincubation with PMA (heterologous desensitization) or LPA (homologous desensitization) on LPA-induced intracellular calcium concentration ([Ca2+]i) using high agonist concentrations.Cells overexpressing LPA1 (panel A, black), LPA2 (panel B, blue) or LPA3 (panel C, red) receptors were preincubated in the absence or presence of 1 μM LPA for 10 min, extensively washed, then challenged with the indicated concentrations of 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 and challenged with 1 μM LPA (C 1 in the abscisa) (% of control) as mean ± S. E. M. of 6 experiments using different cell preparations. In the right panels concentration response curves are plotted. Data from Fig 1 (without pre-stimulation) were normalized and re-plotted as percentage of the maximal response (solid symbols and continuous connecting lines) together with those in the left panels of this figure, normalized in the same way (open symbols, dotted connected lines). The response of cells preincubated with 1 μM PMA for 2 min, washed, and then challenged with to 100 μM LPA is also presented (solid triangles).

Mentions: In order to evaluate heterologous desensitization (PMA-induced) cells were incubated with different concentrations of PMA for 2 min and then challenged with 1 μM LPA. As illustrated in Fig 2 (panel A), cells overexpressing LPA1 receptors were very sensitive to PMA (IC50 1–3 nM) whereas those overexpressing LPA2 and LPA3 receptors were slightly less sensitive (IC50 values in the range of 3–10 nM). Interestingly, in LPA2-overexpressing cells PMA was unable to completely blunt the calcium response to the bioactive lipid, i. e., a remaining ~ 30–40% response was consistently observed, even at the highest PMA concentration tested (Fig 2, panel A; representative tracings are presented in “Fig B in S1 File”). In the case of LPA-induced (homologous) desensitization, cells expressing the different LPA receptors were incubated for 10 min in the presence of different LPA concentrations; after this, the cells were washed twice to remove LPA and were then challenged with 1 μM LPA. The results showed that cells expressing any of the three receptors were affected by the preincubation, even at very low concentrations of the agonist (Fig 2, panel B); the preincubation and washing procedures were not responsible for this as evidenced by the control responses (baseline, vehicle during the preincubation). Agonists-mediated decreases occurred in a concentration-dependent fashion, with a maximum at the concentration of ~ 100 nM. Interestingly, the magnitude of this desensitization process was LPA1 ≥LPA3> LPA2 (Fig 2, panel B). When cells were incubated for 10 min with 1 μM LPA, washed as indicated above and then challenged with 1–100 μM LPA the calcium response increased. This indicated that rather than decreasing the maximal response, homologous desensitization reduced the cell’s sensitivity to LPA (Fig 3, left panels). This was more clearly shown when the concentration-response curves to LPA in control and agonist-pretreated cells were normalized and plotted (Fig 3, right panels). It is worth noticing that the shift in the curves was more pronounced in LPA1- or LPA3-expressing cells, than in those that expressed the LPA2 subtype. The response to 100 μM LPA of PMA-treated cells was very small (Fig 3, right panels).


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

Alcántara-Hernández R, Hernández-Méndez A, Campos-Martínez GA, Meizoso-Huesca A, García-Sáinz JA - PLoS ONE (2015)

Effect of preincubation with PMA (heterologous desensitization) or LPA (homologous desensitization) on LPA-induced intracellular calcium concentration ([Ca2+]i) using high agonist concentrations.Cells overexpressing LPA1 (panel A, black), LPA2 (panel B, blue) or LPA3 (panel C, red) receptors were preincubated in the absence or presence of 1 μM LPA for 10 min, extensively washed, then challenged with the indicated concentrations of 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 and challenged with 1 μM LPA (C 1 in the abscisa) (% of control) as mean ± S. E. M. of 6 experiments using different cell preparations. In the right panels concentration response curves are plotted. Data from Fig 1 (without pre-stimulation) were normalized and re-plotted as percentage of the maximal response (solid symbols and continuous connecting lines) together with those in the left panels of this figure, normalized in the same way (open symbols, dotted connected lines). The response of cells preincubated with 1 μM PMA for 2 min, washed, and then challenged with to 100 μM LPA is also presented (solid triangles).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4608732&req=5

pone.0140583.g003: Effect of preincubation with PMA (heterologous desensitization) or LPA (homologous desensitization) on LPA-induced intracellular calcium concentration ([Ca2+]i) using high agonist concentrations.Cells overexpressing LPA1 (panel A, black), LPA2 (panel B, blue) or LPA3 (panel C, red) receptors were preincubated in the absence or presence of 1 μM LPA for 10 min, extensively washed, then challenged with the indicated concentrations of 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 and challenged with 1 μM LPA (C 1 in the abscisa) (% of control) as mean ± S. E. M. of 6 experiments using different cell preparations. In the right panels concentration response curves are plotted. Data from Fig 1 (without pre-stimulation) were normalized and re-plotted as percentage of the maximal response (solid symbols and continuous connecting lines) together with those in the left panels of this figure, normalized in the same way (open symbols, dotted connected lines). The response of cells preincubated with 1 μM PMA for 2 min, washed, and then challenged with to 100 μM LPA is also presented (solid triangles).
Mentions: In order to evaluate heterologous desensitization (PMA-induced) cells were incubated with different concentrations of PMA for 2 min and then challenged with 1 μM LPA. As illustrated in Fig 2 (panel A), cells overexpressing LPA1 receptors were very sensitive to PMA (IC50 1–3 nM) whereas those overexpressing LPA2 and LPA3 receptors were slightly less sensitive (IC50 values in the range of 3–10 nM). Interestingly, in LPA2-overexpressing cells PMA was unable to completely blunt the calcium response to the bioactive lipid, i. e., a remaining ~ 30–40% response was consistently observed, even at the highest PMA concentration tested (Fig 2, panel A; representative tracings are presented in “Fig B in S1 File”). In the case of LPA-induced (homologous) desensitization, cells expressing the different LPA receptors were incubated for 10 min in the presence of different LPA concentrations; after this, the cells were washed twice to remove LPA and were then challenged with 1 μM LPA. The results showed that cells expressing any of the three receptors were affected by the preincubation, even at very low concentrations of the agonist (Fig 2, panel B); the preincubation and washing procedures were not responsible for this as evidenced by the control responses (baseline, vehicle during the preincubation). Agonists-mediated decreases occurred in a concentration-dependent fashion, with a maximum at the concentration of ~ 100 nM. Interestingly, the magnitude of this desensitization process was LPA1 ≥LPA3> LPA2 (Fig 2, panel B). When cells were incubated for 10 min with 1 μM LPA, washed as indicated above and then challenged with 1–100 μM LPA the calcium response increased. This indicated that rather than decreasing the maximal response, homologous desensitization reduced the cell’s sensitivity to LPA (Fig 3, left panels). This was more clearly shown when the concentration-response curves to LPA in control and agonist-pretreated cells were normalized and plotted (Fig 3, right panels). It is worth noticing that the shift in the curves was more pronounced in LPA1- or LPA3-expressing cells, than in those that expressed the LPA2 subtype. The response to 100 μM LPA of PMA-treated cells was very small (Fig 3, right panels).

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Apartado Postal 70-248, México D.F., México 04510.

ABSTRACT

Results: 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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus