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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.


Effect of PKC down regulation on heterologous (PMA-induced) or homologous (LPA-induced) desensitization.Cells overexpressing LPA1–3 receptors were preincubated overnight with 1 μM PMA, and then subjected to the desensitization protocols (indicated under the Experimental section and in Fig 2) 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.
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pone.0140583.g006: Effect of PKC down regulation on heterologous (PMA-induced) or homologous (LPA-induced) desensitization.Cells overexpressing LPA1–3 receptors were preincubated overnight with 1 μM PMA, and then subjected to the desensitization protocols (indicated under the Experimental section and in Fig 2) 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.

Mentions: It is well-known that over-night treatment with 1 μM PMA markedly down-regulates conventional and novel PKC isoforms [14, 28]. Consistent with the previous data, this treatment markedly reduced or abolished PMA-induced desensitization (Fig 6, panel A) but was completely unable to alter agonist-induced desensitization (Fig 6, panel B).


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 PKC down regulation on heterologous (PMA-induced) or homologous (LPA-induced) desensitization.Cells overexpressing LPA1–3 receptors were preincubated overnight with 1 μM PMA, and then subjected to the desensitization protocols (indicated under the Experimental section and in Fig 2) 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.
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Related In: Results  -  Collection

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pone.0140583.g006: Effect of PKC down regulation on heterologous (PMA-induced) or homologous (LPA-induced) desensitization.Cells overexpressing LPA1–3 receptors were preincubated overnight with 1 μM PMA, and then subjected to the desensitization protocols (indicated under the Experimental section and in Fig 2) 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.
Mentions: It is well-known that over-night treatment with 1 μM PMA markedly down-regulates conventional and novel PKC isoforms [14, 28]. Consistent with the previous data, this treatment markedly reduced or abolished PMA-induced desensitization (Fig 6, panel A) but was completely unable to alter agonist-induced desensitization (Fig 6, panel B).

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.