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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

Transactivation of EGF receptors in LPA-induced ERK 1/2 phosphorylation.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.
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pone.0140583.g008: Transactivation of EGF receptors in LPA-induced ERK 1/2 phosphorylation.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.

Mentions: We next examined another further downstream functional response: ERK phosphorylation. Phosphorylation of this key enzyme has been observed in response to LPA1–3 receptor activation [29–32]. As shown in Fig 7, activation of any of the three receptors studied was able to activate ERK as reflected by its phosphorylation state. The magnitude of the response was somewhat different, with that induced by LPA1 receptors greater and with a more prolonged duration than that induced by LPA3 receptor activation and that due to LPA2 activation was clearly smaller and lesser in duration (Fig 7). Elegant work by Ullrich and coworkers has shown that many GPCRs, including LPA receptors (subtype(s) not defined) can transactivate EGF receptors, through sequential metalloproteinase activation and HB-EGF shedding and that joint signaling through GPCRs and the EGF tyrosine kinase activity participates in some of the actions ([33] reviewed in [34–37]). Previously, we showed that activation LPA1 receptors induce Akt/PKB phosphorylation through the previously-mentioned EGF receptor transactivation process [12]. In the present experiments, the possible role of EGF receptor transactivation was evaluated, by inhibiting the EGF receptor kinase with the selective tyrphostin, AG1478 [38]. As presented in Fig 6, both LPA (1 μM) and EGF (100 ng/ml) increase ERK phosphorylation (~ 2- and ~ 4-fold, respectively). AG1478 clearly diminished the effect of both growth factors and, in some cases, to below the baseline signal (Fig 8). The baseline phospho-ERK signal was very low and AG1478 either did not alter it (cells expressing LPA1 receptors) or decrease it (cells overexpressing LPA2 (statistically insignificant) and LPA3 receptors (statistically significant) (“Fig D 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)

Transactivation of EGF receptors in LPA-induced ERK 1/2 phosphorylation.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.
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pone.0140583.g008: Transactivation of EGF receptors in LPA-induced ERK 1/2 phosphorylation.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.
Mentions: We next examined another further downstream functional response: ERK phosphorylation. Phosphorylation of this key enzyme has been observed in response to LPA1–3 receptor activation [29–32]. As shown in Fig 7, activation of any of the three receptors studied was able to activate ERK as reflected by its phosphorylation state. The magnitude of the response was somewhat different, with that induced by LPA1 receptors greater and with a more prolonged duration than that induced by LPA3 receptor activation and that due to LPA2 activation was clearly smaller and lesser in duration (Fig 7). Elegant work by Ullrich and coworkers has shown that many GPCRs, including LPA receptors (subtype(s) not defined) can transactivate EGF receptors, through sequential metalloproteinase activation and HB-EGF shedding and that joint signaling through GPCRs and the EGF tyrosine kinase activity participates in some of the actions ([33] reviewed in [34–37]). Previously, we showed that activation LPA1 receptors induce Akt/PKB phosphorylation through the previously-mentioned EGF receptor transactivation process [12]. In the present experiments, the possible role of EGF receptor transactivation was evaluated, by inhibiting the EGF receptor kinase with the selective tyrphostin, AG1478 [38]. As presented in Fig 6, both LPA (1 μM) and EGF (100 ng/ml) increase ERK phosphorylation (~ 2- and ~ 4-fold, respectively). AG1478 clearly diminished the effect of both growth factors and, in some cases, to below the baseline signal (Fig 8). The baseline phospho-ERK signal was very low and AG1478 either did not alter it (cells expressing LPA1 receptors) or decrease it (cells overexpressing LPA2 (statistically insignificant) and LPA3 receptors (statistically significant) (“Fig D 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.


Related in: MedlinePlus