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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 LPA on intracellular calcium concentration ([Ca2+]i).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.
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pone.0140583.g001: Effect of LPA on intracellular calcium concentration ([Ca2+]i).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.

Mentions: In agreement with previous results [10, 11] wild-type C9 cells increase intracellular calcium concentration in response to LPA; this is likely due to the activation of LPA1 and LPA2 that are expressed in these cells [11]. The effect was concentration-dependent with a maximal calcium concentration increase of ~150–200 nM and EC50 values in the range of ~ 200–400 nM (Fig 1, panel A). Expression of LPA1–3 receptors markedly augmented this calcium response to increases the cation’s concentration to ~ 300 nM (in cells over-expressing LPA3 receptors) and to 450–600 nM (cells over-expressing LPA1 or LPA2 receptors); concentration-response curves yielded EC50 values in the range of 200–400 nM in LPA1- or LPA3- overexpressing cells. In cells overexpressing LPA2, the LPA concentration response curve was slightly, but consistently, shifted to the right and no clear saturation was achieved at the concentrations tested (Fig 1, panels B-D; representative images in “Fig B in S1 File”).


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 LPA on intracellular calcium concentration ([Ca2+]i).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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140583.g001: Effect of LPA on intracellular calcium concentration ([Ca2+]i).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.
Mentions: In agreement with previous results [10, 11] wild-type C9 cells increase intracellular calcium concentration in response to LPA; this is likely due to the activation of LPA1 and LPA2 that are expressed in these cells [11]. The effect was concentration-dependent with a maximal calcium concentration increase of ~150–200 nM and EC50 values in the range of ~ 200–400 nM (Fig 1, panel A). Expression of LPA1–3 receptors markedly augmented this calcium response to increases the cation’s concentration to ~ 300 nM (in cells over-expressing LPA3 receptors) and to 450–600 nM (cells over-expressing LPA1 or LPA2 receptors); concentration-response curves yielded EC50 values in the range of 200–400 nM in LPA1- or LPA3- overexpressing cells. In cells overexpressing LPA2, the LPA concentration response curve was slightly, but consistently, shifted to the right and no clear saturation was achieved at the concentrations tested (Fig 1, panels B-D; representative images in “Fig B in S1 File”).

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.