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.


Related in: MedlinePlus

Images of the effects of LPA and PMA on LPA1–3 receptor internalization (60 and 120 min).Fluorescent confocal images of cells overexpressing LPA1 (column A), LPA2 (column B) or LPA3 (column C) receptors. Cells were incubated in the absence of any agent (Baseline), for 10 min in the presence of 1 μM LPA, or for 2 min in the presence of 1 μM PMA. After this incubation cells were extensively washed and further incubated for the times indicated. Images are representative of data of 3 experiments using different cell preparations. Bars 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140583.g015: Images of the effects of LPA and PMA on LPA1–3 receptor internalization (60 and 120 min).Fluorescent confocal images of cells overexpressing LPA1 (column A), LPA2 (column B) or LPA3 (column C) receptors. Cells were incubated in the absence of any agent (Baseline), for 10 min in the presence of 1 μM LPA, or for 2 min in the presence of 1 μM PMA. After this incubation cells were extensively washed and further incubated for the times indicated. Images are representative of data of 3 experiments using different cell preparations. Bars 10 μm.

Mentions: It has been observed that EGF receptor transactivation plays a role in the phosphorylation of some GPCRs, such as the α1B-adrenergic receptor [39–42]. As shown in Fig 11, this was also the case in agonist-induced LPA1–3 receptor phosphorylation, i. e., the EGF receptor tyrosine kinase inhibitor, AG1478, markedly reduced (but did not abolish) LPA-induced phosphorylation of the three receptor subtypes studied. The inhibitor by itself did not alter basal receptor phosphorylation (data not shown). As already mentioned, prolonged treatment (over-night) with 1 μM PMA markedly down-regulates conventional and novel PKC isoforms [14, 28]. Consistent with previous data on LPA1 receptors [14], this treatment markedly reduced or abolished PMA-induced LPA1–3 phosphorylation but did not alter LPA-induced receptor phosphorylation (Fig 12). GPCR phosphorylation appears to be associated with receptor internalization. Current ideas suggest that GPCR phosphorylation increases the receptor’s affinity for β-arrestins, which associates with clathrin favoring the formation of receptor-enriched coated pits, triggering internalization [43–46]. As depicted in the confocal images presented as in Fig 13, fluorescence (i. e. the eGFP-tagged receptors) was present in both, the plasma membrane and intracellular vesicles. Treatment with LPA or PMA clearly altered receptor distribution, markedly decreasing fluorescence at the plasma membrane level and increasing that in intracellular vesicles. Such changes were clearly observed only after 20–30 min indicating that desensitization precedes internalization. Differences were observed among the LPA receptor subtypes after continuous exposure to these agents for 30 or 60 min. The decrease in fluorescence at the plasma membrane was much less intense in LPA2-overexpressing cells as compared with that in those overexpressing the other subtypes (Fig 13) (see also images overlapping fluorescence and differential interference contrast in “Fig E in S1 File”); quantitative analysis also clearly evidenced this (Fig 14). Differences were also observed in the morphology (small punctuated or large vesicles) and localization (concentrated in perinuclear region or distributed in the whole cell) of the internalized fluorescence both among the distinct receptors studied and also depending on the stimulus. Analysis of such differences will require a systematic work with different approaches. In an effort to get further insight into the receptor traffic dynamics, images were obtained in cells treated with 1 μM LPA (10 min), exhaustively washed, and further incubated to complete 60 or 120 min of incubation (Fig 15). It was observed that under these conditions fluorescence recovered rapidly and completely at the membrane level in cells expressing LPA1 receptors; slowly in cells expressing LPA2 receptors, and only partially and also slowly in cells expressing LPA3 receptors (Fig 15, see also “Fig F in S1 File”); quantitative analysis is presented in Fig 16. Fluorescence recovery at the plasma membrane could represent, receptor recycling, incorporation of receptors previously present in vesicles and newly synthesized ones, and, more likely a mixture of all these. Distinction among such processes will also require a systematic work with different approaches.


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)

Images of the effects of LPA and PMA on LPA1–3 receptor internalization (60 and 120 min).Fluorescent confocal images of cells overexpressing LPA1 (column A), LPA2 (column B) or LPA3 (column C) receptors. Cells were incubated in the absence of any agent (Baseline), for 10 min in the presence of 1 μM LPA, or for 2 min in the presence of 1 μM PMA. After this incubation cells were extensively washed and further incubated for the times indicated. Images are representative of data of 3 experiments using different cell preparations. Bars 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140583.g015: Images of the effects of LPA and PMA on LPA1–3 receptor internalization (60 and 120 min).Fluorescent confocal images of cells overexpressing LPA1 (column A), LPA2 (column B) or LPA3 (column C) receptors. Cells were incubated in the absence of any agent (Baseline), for 10 min in the presence of 1 μM LPA, or for 2 min in the presence of 1 μM PMA. After this incubation cells were extensively washed and further incubated for the times indicated. Images are representative of data of 3 experiments using different cell preparations. Bars 10 μm.
Mentions: It has been observed that EGF receptor transactivation plays a role in the phosphorylation of some GPCRs, such as the α1B-adrenergic receptor [39–42]. As shown in Fig 11, this was also the case in agonist-induced LPA1–3 receptor phosphorylation, i. e., the EGF receptor tyrosine kinase inhibitor, AG1478, markedly reduced (but did not abolish) LPA-induced phosphorylation of the three receptor subtypes studied. The inhibitor by itself did not alter basal receptor phosphorylation (data not shown). As already mentioned, prolonged treatment (over-night) with 1 μM PMA markedly down-regulates conventional and novel PKC isoforms [14, 28]. Consistent with previous data on LPA1 receptors [14], this treatment markedly reduced or abolished PMA-induced LPA1–3 phosphorylation but did not alter LPA-induced receptor phosphorylation (Fig 12). GPCR phosphorylation appears to be associated with receptor internalization. Current ideas suggest that GPCR phosphorylation increases the receptor’s affinity for β-arrestins, which associates with clathrin favoring the formation of receptor-enriched coated pits, triggering internalization [43–46]. As depicted in the confocal images presented as in Fig 13, fluorescence (i. e. the eGFP-tagged receptors) was present in both, the plasma membrane and intracellular vesicles. Treatment with LPA or PMA clearly altered receptor distribution, markedly decreasing fluorescence at the plasma membrane level and increasing that in intracellular vesicles. Such changes were clearly observed only after 20–30 min indicating that desensitization precedes internalization. Differences were observed among the LPA receptor subtypes after continuous exposure to these agents for 30 or 60 min. The decrease in fluorescence at the plasma membrane was much less intense in LPA2-overexpressing cells as compared with that in those overexpressing the other subtypes (Fig 13) (see also images overlapping fluorescence and differential interference contrast in “Fig E in S1 File”); quantitative analysis also clearly evidenced this (Fig 14). Differences were also observed in the morphology (small punctuated or large vesicles) and localization (concentrated in perinuclear region or distributed in the whole cell) of the internalized fluorescence both among the distinct receptors studied and also depending on the stimulus. Analysis of such differences will require a systematic work with different approaches. In an effort to get further insight into the receptor traffic dynamics, images were obtained in cells treated with 1 μM LPA (10 min), exhaustively washed, and further incubated to complete 60 or 120 min of incubation (Fig 15). It was observed that under these conditions fluorescence recovered rapidly and completely at the membrane level in cells expressing LPA1 receptors; slowly in cells expressing LPA2 receptors, and only partially and also slowly in cells expressing LPA3 receptors (Fig 15, see also “Fig F in S1 File”); quantitative analysis is presented in Fig 16. Fluorescence recovery at the plasma membrane could represent, receptor recycling, incorporation of receptors previously present in vesicles and newly synthesized ones, and, more likely a mixture of all these. Distinction among such processes will also require a systematic work with different approaches.

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