Limits...
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.


Time-courses of the effects of LPA and PMA on LPA1–3 receptor phosphorylation.Cells overexpressing LPA1 (black, circles), LPA2 (blue, squares) or LPA3 (red, triangles) receptors were incubated for the times indicated in the presence of 1 μM LPA (Panel A) or 1 μM PMA (Panel B). Plotted are the percentage of baseline phosphorylations as mean ± S. E. M. of 4–5 experiments using different cell preparations. Representative autoradiographs are presented for the different receptor subtypes.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4608732&req=5

pone.0140583.g009: Time-courses of the effects of LPA and PMA on LPA1–3 receptor phosphorylation.Cells overexpressing LPA1 (black, circles), LPA2 (blue, squares) or LPA3 (red, triangles) receptors were incubated for the times indicated in the presence of 1 μM LPA (Panel A) or 1 μM PMA (Panel B). Plotted are the percentage of baseline phosphorylations as mean ± S. E. M. of 4–5 experiments using different cell preparations. Representative autoradiographs are presented for the different receptor subtypes.

Mentions: Agonist- and PMA-induced receptor phosphorylation was examined next. Data showed that the three LPA receptors studied are phosphoproteins whose phosphorylation states are increased by LPA and the active phorbol ester, PMA (Figs 9 and 10). The time-course of the effect of 1 μM LPA (Fig 9, panel A) showed that in cells expressing any of the receptors studied, the agonist increased receptor phosphorylation, and this reached its maximum during the first 15 min and remained at the same level for up to 60 min. The effect of 1 μM PMA (Fig 9, panel B) took place faster than that of the agonist, reaching its maximum during the first 5 min and remaining at a plateau during the time studied (60 min). Interestingly, the relative magnitudes (percentage of baseline labeling) and temporal patterns were similar for all three receptor subtypes, although in some experiments LPA2 receptor phosphorylation was slightly delayed. The concentration-response curves to LPA and PMA are presented in Fig 10 (panels A and B, respectively). It can be observed that the curves for LPA and PMA were very similar for LPA1- and LPA3-overexpressing cells; saturation was obtained at ~ 1 μM LPA (EC50 10–30 nM) and 100 nM PMA (EC50 ~ 3–10 nM) (Fig 10). In the studies utilizing cells that overexpress LPA2 receptors, no clear saturation was obtained for either LPA or PMA (Fig 10; panels A and B, respectively); under these conditions EC50 values could not be estimated, but the concentration-response curves exhibited a clear shift to higher concentrations as compared with those studying the remaining receptor subtypes.


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)

Time-courses of the effects of LPA and PMA on LPA1–3 receptor phosphorylation.Cells overexpressing LPA1 (black, circles), LPA2 (blue, squares) or LPA3 (red, triangles) receptors were incubated for the times indicated in the presence of 1 μM LPA (Panel A) or 1 μM PMA (Panel B). Plotted are the percentage of baseline phosphorylations as mean ± S. E. M. of 4–5 experiments using different cell preparations. Representative autoradiographs are presented for the different receptor subtypes.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140583.g009: Time-courses of the effects of LPA and PMA on LPA1–3 receptor phosphorylation.Cells overexpressing LPA1 (black, circles), LPA2 (blue, squares) or LPA3 (red, triangles) receptors were incubated for the times indicated in the presence of 1 μM LPA (Panel A) or 1 μM PMA (Panel B). Plotted are the percentage of baseline phosphorylations as mean ± S. E. M. of 4–5 experiments using different cell preparations. Representative autoradiographs are presented for the different receptor subtypes.
Mentions: Agonist- and PMA-induced receptor phosphorylation was examined next. Data showed that the three LPA receptors studied are phosphoproteins whose phosphorylation states are increased by LPA and the active phorbol ester, PMA (Figs 9 and 10). The time-course of the effect of 1 μM LPA (Fig 9, panel A) showed that in cells expressing any of the receptors studied, the agonist increased receptor phosphorylation, and this reached its maximum during the first 15 min and remained at the same level for up to 60 min. The effect of 1 μM PMA (Fig 9, panel B) took place faster than that of the agonist, reaching its maximum during the first 5 min and remaining at a plateau during the time studied (60 min). Interestingly, the relative magnitudes (percentage of baseline labeling) and temporal patterns were similar for all three receptor subtypes, although in some experiments LPA2 receptor phosphorylation was slightly delayed. The concentration-response curves to LPA and PMA are presented in Fig 10 (panels A and B, respectively). It can be observed that the curves for LPA and PMA were very similar for LPA1- and LPA3-overexpressing cells; saturation was obtained at ~ 1 μM LPA (EC50 10–30 nM) and 100 nM PMA (EC50 ~ 3–10 nM) (Fig 10). In the studies utilizing cells that overexpress LPA2 receptors, no clear saturation was obtained for either LPA or PMA (Fig 10; panels A and B, respectively); under these conditions EC50 values could not be estimated, but the concentration-response curves exhibited a clear shift to higher concentrations as compared with those studying the remaining receptor subtypes.

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.