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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.


Reversibility (homologous) and persistency (heterologous) of the desensitizations of the intracellular calcium response to LPA.Cells overexpressing LPA1 (panel A, black symbols and lines), LPA2 (panel B, blue symbols and lines) or LPA3 (panel C, red symbols and lines) receptors were preincubated in the presence of 1 μM PMA for 2 min (open symbols, dotted lines) or with 1 μM LPA for 10 minutes and then extensively washed. Incubation was continued for the times indicated and 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 (control, time 0) as mean ± S. E. M. of 5–6 experiments using different cell preparations.
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pone.0140583.g004: Reversibility (homologous) and persistency (heterologous) of the desensitizations of the intracellular calcium response to LPA.Cells overexpressing LPA1 (panel A, black symbols and lines), LPA2 (panel B, blue symbols and lines) or LPA3 (panel C, red symbols and lines) receptors were preincubated in the presence of 1 μM PMA for 2 min (open symbols, dotted lines) or with 1 μM LPA for 10 minutes and then extensively washed. Incubation was continued for the times indicated and 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 (control, time 0) as mean ± S. E. M. of 5–6 experiments using different cell preparations.

Mentions: The possibility that the cell responsiveness to LPA could resensitize was considered. To study this, cells were incubated with 1 μM PMA for 2 min or with 1 μM LPA for 10 min and then extensively washed. After this procedure, the incubation continued for the times indicated (up to 2 h) and then cells were challenged with 1 μM LPA. As shown in Fig 4 the cell responsiveness to LPA recover after washing, during the subsequent incubation but not when the active phorbol ester, PMA, was employed. These resensitization patterns were similar for the three LPA receptor subtypes studied (Fig 4). The resenstization time-courses after homologous desensitization were inversely correlated with the desensitization magnitudes, i. e., cells expressing LPA2.receptors resensitize clearly faster that those expressing the LPA1 or LPA3 subtypes (Fig 4). We were unable to detect changes in receptor density (degradation) in response to LPA or PMA during these incubations times, as evidenced by anti-eGFP Western blotting of extracts from cells incubated in the presence cycloheximide (50 μM) to prevent new protein synthesis; cycloheximide was added 30 min before addition of LPA or PMA and was present during the whole incubation period (“Fig C 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)

Reversibility (homologous) and persistency (heterologous) of the desensitizations of the intracellular calcium response to LPA.Cells overexpressing LPA1 (panel A, black symbols and lines), LPA2 (panel B, blue symbols and lines) or LPA3 (panel C, red symbols and lines) receptors were preincubated in the presence of 1 μM PMA for 2 min (open symbols, dotted lines) or with 1 μM LPA for 10 minutes and then extensively washed. Incubation was continued for the times indicated and 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 (control, time 0) as mean ± S. E. M. of 5–6 experiments using different cell preparations.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140583.g004: Reversibility (homologous) and persistency (heterologous) of the desensitizations of the intracellular calcium response to LPA.Cells overexpressing LPA1 (panel A, black symbols and lines), LPA2 (panel B, blue symbols and lines) or LPA3 (panel C, red symbols and lines) receptors were preincubated in the presence of 1 μM PMA for 2 min (open symbols, dotted lines) or with 1 μM LPA for 10 minutes and then extensively washed. Incubation was continued for the times indicated and 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 (control, time 0) as mean ± S. E. M. of 5–6 experiments using different cell preparations.
Mentions: The possibility that the cell responsiveness to LPA could resensitize was considered. To study this, cells were incubated with 1 μM PMA for 2 min or with 1 μM LPA for 10 min and then extensively washed. After this procedure, the incubation continued for the times indicated (up to 2 h) and then cells were challenged with 1 μM LPA. As shown in Fig 4 the cell responsiveness to LPA recover after washing, during the subsequent incubation but not when the active phorbol ester, PMA, was employed. These resensitization patterns were similar for the three LPA receptor subtypes studied (Fig 4). The resenstization time-courses after homologous desensitization were inversely correlated with the desensitization magnitudes, i. e., cells expressing LPA2.receptors resensitize clearly faster that those expressing the LPA1 or LPA3 subtypes (Fig 4). We were unable to detect changes in receptor density (degradation) in response to LPA or PMA during these incubations times, as evidenced by anti-eGFP Western blotting of extracts from cells incubated in the presence cycloheximide (50 μM) to prevent new protein synthesis; cycloheximide was added 30 min before addition of LPA or PMA and was present during the whole incubation period (“Fig C 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.