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Phosphatidic acid induces ligand-independent epidermal growth factor receptor endocytic traffic through PDE4 activation.

Norambuena A, Metz C, Jung JE, Silva A, Otero C, Cancino J, Retamal C, Valenzuela JC, Soza A, González A - Mol. Biol. Cell (2010)

Bottom Line: Accelerated EGFR endocytosis seems to be mediated by clathrin-dependent and -independent pathways, leading to receptor accumulation in juxtanuclear recycling endosomes, also due to a decreased recycling.Internalized EGFR can remain intracellular without degradation for several hours or return rapidly to the cell surface upon discontinuation of the stimulus.This novel regulatory mechanism of EGFR, also novel function of signaling PA, can transmodulate receptor accessibility in response to heterologous stimuli.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Inmunología Clínica y Reumatología, Facultad de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile.

ABSTRACT
Endocytosis modulates EGFR function by compartmentalizing and attenuating or enhancing its ligand-induced signaling. Here we show that it can also control the cell surface versus intracellular distribution of empty/inactive EGFR. Our previous observation that PKA inhibitors induce EGFR internalization prompted us to test phosphatidic acid (PA) generated by phospholipase D (PLD) as an endogenous down-regulator of PKA activity, which activates rolipram-sensitive type 4 phosphodiesterases (PDE4) that degrade cAMP. We found that inhibition of PA hydrolysis by propranolol, in the absence of ligand, provokes internalization of inactive (neither tyrosine-phosphorylated nor ubiquitinated) EGFR, accompanied by a transient increase in PA levels and PDE4s activity. This EGFR internalization is mimicked by PA micelles and is strongly counteracted by PLD2 silencing, rolipram or forskolin treatment, and PKA overexpression. Accelerated EGFR endocytosis seems to be mediated by clathrin-dependent and -independent pathways, leading to receptor accumulation in juxtanuclear recycling endosomes, also due to a decreased recycling. Internalized EGFR can remain intracellular without degradation for several hours or return rapidly to the cell surface upon discontinuation of the stimulus. This novel regulatory mechanism of EGFR, also novel function of signaling PA, can transmodulate receptor accessibility in response to heterologous stimuli.

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Propranolol does not affect endocytosis of TfR and μ-opioid receptors, but promotes intracellular accumulation of Tf-Alexa 549. (A) Constitutive endocytosis of TfR. HeLa cells were preincubated in serum-free media for 1 h at 37°C and then with 20 ng/ml 125I-diTf for 2 h at 4°C. After eliminating the unbound 125I-diTf, cells were incubated in the presence or absence of 75 μM propranolol for 5–10 min at 37°C. IN/SUR plot shows no difference in the internalization rates (ke). (B) Propranolol increases the accumulation of Tf-Alexa 549 in juxtanuclear endosomes. Cells incubated with 50 μg/ml Tf-Alexa 549 at 37°C for 30 min in the absence or presence of 75 μM and fixed for fluorescence imaging show strong juxtanuclear fluorescence accumulation, suggesting decreased TfR recycling. (C) Propranolol treatment does not cause redistribution of μ-opioid receptors. N2a cells permanently transfected with either EGFR or FLAG-tagged μ-opioid receptors were incubated with either 75 μM propranolol or 10 μM DAMGO (μ-opioid receptor ligand), for 30 min. Bar, 10 μm.
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Figure 5: Propranolol does not affect endocytosis of TfR and μ-opioid receptors, but promotes intracellular accumulation of Tf-Alexa 549. (A) Constitutive endocytosis of TfR. HeLa cells were preincubated in serum-free media for 1 h at 37°C and then with 20 ng/ml 125I-diTf for 2 h at 4°C. After eliminating the unbound 125I-diTf, cells were incubated in the presence or absence of 75 μM propranolol for 5–10 min at 37°C. IN/SUR plot shows no difference in the internalization rates (ke). (B) Propranolol increases the accumulation of Tf-Alexa 549 in juxtanuclear endosomes. Cells incubated with 50 μg/ml Tf-Alexa 549 at 37°C for 30 min in the absence or presence of 75 μM and fixed for fluorescence imaging show strong juxtanuclear fluorescence accumulation, suggesting decreased TfR recycling. (C) Propranolol treatment does not cause redistribution of μ-opioid receptors. N2a cells permanently transfected with either EGFR or FLAG-tagged μ-opioid receptors were incubated with either 75 μM propranolol or 10 μM DAMGO (μ-opioid receptor ligand), for 30 min. Bar, 10 μm.

Mentions: The effect of PKA inhibitors that induce EGFR internalization is selective (Salazar and Gonzalez, 2002); e.g., neither affect the internalization rate of Tf, which is constitutively endocytosed via the clathrin pathway (Maxfield and McGraw, 2004), nor the distribution of the μ-opioid receptor, which is also endocytosed by the clathrin pathway but in response to agonist (DAMGO) stimulation (Keith et al., 1996). IN/SUR analysis (Wiley and Cunningham, 1982) of 125I-Tf endocytosis showed an endocytic constant (ke) of 0.12 ± 0.013 min−1 that did not significantly change in propranolol-treated cells (ke + 0.19 ± 0.034 min−1; Figure 5A). However, when cells were incubated with Tf-Alexa594 for longer times, 30–60 min, fluorescence was seen accumulated in juxtanuclear recycling compartments, suggesting that propranolol decreases the recycling phase of TfR (Figure 5B).


Phosphatidic acid induces ligand-independent epidermal growth factor receptor endocytic traffic through PDE4 activation.

Norambuena A, Metz C, Jung JE, Silva A, Otero C, Cancino J, Retamal C, Valenzuela JC, Soza A, González A - Mol. Biol. Cell (2010)

Propranolol does not affect endocytosis of TfR and μ-opioid receptors, but promotes intracellular accumulation of Tf-Alexa 549. (A) Constitutive endocytosis of TfR. HeLa cells were preincubated in serum-free media for 1 h at 37°C and then with 20 ng/ml 125I-diTf for 2 h at 4°C. After eliminating the unbound 125I-diTf, cells were incubated in the presence or absence of 75 μM propranolol for 5–10 min at 37°C. IN/SUR plot shows no difference in the internalization rates (ke). (B) Propranolol increases the accumulation of Tf-Alexa 549 in juxtanuclear endosomes. Cells incubated with 50 μg/ml Tf-Alexa 549 at 37°C for 30 min in the absence or presence of 75 μM and fixed for fluorescence imaging show strong juxtanuclear fluorescence accumulation, suggesting decreased TfR recycling. (C) Propranolol treatment does not cause redistribution of μ-opioid receptors. N2a cells permanently transfected with either EGFR or FLAG-tagged μ-opioid receptors were incubated with either 75 μM propranolol or 10 μM DAMGO (μ-opioid receptor ligand), for 30 min. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 5: Propranolol does not affect endocytosis of TfR and μ-opioid receptors, but promotes intracellular accumulation of Tf-Alexa 549. (A) Constitutive endocytosis of TfR. HeLa cells were preincubated in serum-free media for 1 h at 37°C and then with 20 ng/ml 125I-diTf for 2 h at 4°C. After eliminating the unbound 125I-diTf, cells were incubated in the presence or absence of 75 μM propranolol for 5–10 min at 37°C. IN/SUR plot shows no difference in the internalization rates (ke). (B) Propranolol increases the accumulation of Tf-Alexa 549 in juxtanuclear endosomes. Cells incubated with 50 μg/ml Tf-Alexa 549 at 37°C for 30 min in the absence or presence of 75 μM and fixed for fluorescence imaging show strong juxtanuclear fluorescence accumulation, suggesting decreased TfR recycling. (C) Propranolol treatment does not cause redistribution of μ-opioid receptors. N2a cells permanently transfected with either EGFR or FLAG-tagged μ-opioid receptors were incubated with either 75 μM propranolol or 10 μM DAMGO (μ-opioid receptor ligand), for 30 min. Bar, 10 μm.
Mentions: The effect of PKA inhibitors that induce EGFR internalization is selective (Salazar and Gonzalez, 2002); e.g., neither affect the internalization rate of Tf, which is constitutively endocytosed via the clathrin pathway (Maxfield and McGraw, 2004), nor the distribution of the μ-opioid receptor, which is also endocytosed by the clathrin pathway but in response to agonist (DAMGO) stimulation (Keith et al., 1996). IN/SUR analysis (Wiley and Cunningham, 1982) of 125I-Tf endocytosis showed an endocytic constant (ke) of 0.12 ± 0.013 min−1 that did not significantly change in propranolol-treated cells (ke + 0.19 ± 0.034 min−1; Figure 5A). However, when cells were incubated with Tf-Alexa594 for longer times, 30–60 min, fluorescence was seen accumulated in juxtanuclear recycling compartments, suggesting that propranolol decreases the recycling phase of TfR (Figure 5B).

Bottom Line: Accelerated EGFR endocytosis seems to be mediated by clathrin-dependent and -independent pathways, leading to receptor accumulation in juxtanuclear recycling endosomes, also due to a decreased recycling.Internalized EGFR can remain intracellular without degradation for several hours or return rapidly to the cell surface upon discontinuation of the stimulus.This novel regulatory mechanism of EGFR, also novel function of signaling PA, can transmodulate receptor accessibility in response to heterologous stimuli.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Inmunología Clínica y Reumatología, Facultad de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile.

ABSTRACT
Endocytosis modulates EGFR function by compartmentalizing and attenuating or enhancing its ligand-induced signaling. Here we show that it can also control the cell surface versus intracellular distribution of empty/inactive EGFR. Our previous observation that PKA inhibitors induce EGFR internalization prompted us to test phosphatidic acid (PA) generated by phospholipase D (PLD) as an endogenous down-regulator of PKA activity, which activates rolipram-sensitive type 4 phosphodiesterases (PDE4) that degrade cAMP. We found that inhibition of PA hydrolysis by propranolol, in the absence of ligand, provokes internalization of inactive (neither tyrosine-phosphorylated nor ubiquitinated) EGFR, accompanied by a transient increase in PA levels and PDE4s activity. This EGFR internalization is mimicked by PA micelles and is strongly counteracted by PLD2 silencing, rolipram or forskolin treatment, and PKA overexpression. Accelerated EGFR endocytosis seems to be mediated by clathrin-dependent and -independent pathways, leading to receptor accumulation in juxtanuclear recycling endosomes, also due to a decreased recycling. Internalized EGFR can remain intracellular without degradation for several hours or return rapidly to the cell surface upon discontinuation of the stimulus. This novel regulatory mechanism of EGFR, also novel function of signaling PA, can transmodulate receptor accessibility in response to heterologous stimuli.

Show MeSH
Related in: MedlinePlus