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Revisiting the endocytosis of the m2 muscarinic acetylcholine receptor.

Ockenga W, Tikkanen R - Membranes (Basel) (2015)

Bottom Line: The uptake of the M2 receptor involves the adapter proteins of the β-arrestin family and the small GTPase ADP-ribosylation factor 6.However, it has remained inconclusive if M2 endocytosis is dependent on clathrin or the large GTPase dynamin.The expression of various dominant-negative dynamin-2 mutants and the use of chemical inhibitors of dynamin function revealed that dynamin expression and membrane localization as such appear to be necessary for M2 endocytosis, whereas dynamin GTPase activity is not required for this process.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Medical Faculty, University of Giessen, Friedrichstrasse 24, D-35392 Giessen, Germany. Wymke.Ockenga@biochemie.med.uni-giessen.de.

ABSTRACT
The agonist-induced endocytosis of the muscarinic acetylcholine receptor M2 is different from that of the other members of the muscarinic receptor family. The uptake of the M2 receptor involves the adapter proteins of the β-arrestin family and the small GTPase ADP-ribosylation factor 6. However, it has remained inconclusive if M2 endocytosis is dependent on clathrin or the large GTPase dynamin. We here show by means of knocking down the clathrin heavy chain that M2 uptake upon agonist stimulation requires clathrin. The expression of various dominant-negative dynamin-2 mutants and the use of chemical inhibitors of dynamin function revealed that dynamin expression and membrane localization as such appear to be necessary for M2 endocytosis, whereas dynamin GTPase activity is not required for this process. Based on the data from the present and from previous studies, we propose that M2 endocytosis takes place by means of an atypical clathrin-mediated pathway that may involve a specific subset of clathrin-coated pits/vesicles.

No MeSH data available.


Related in: MedlinePlus

Knockdown of flotillin-1 and flotillin-2 has no impact on M2 receptor internalization. (A) HEK 293T cells were depleted of flotillin-1 (F1), flotillin-2 (F2) or both (F1 + F2) by specific siRNAs and transfected with M2-EGFP. The cells were starved for 18 h in serum-free medium and either left untreated or stimulated with 1 mM CCh for 15 min. Cells were fixed and immunostained for flotillin-1 and flotillin-2. The nuclei were stained with DAPI (shown in blue). Scale bars: 10 μm; (B) The fraction of cells displaying M2 receptor internalization was quantified and is shown as the percent of cells showing intracellular M2 localization. At least 100 cells were counted per condition. Results are shown as the mean ± SD. Statistical analysis was performed with two-way ANOVA; (C) Equal amounts of cell lysates were separated by SDS-PAGE and immunoblotted to monitor the knockdown efficiency.
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membranes-05-00197-f003: Knockdown of flotillin-1 and flotillin-2 has no impact on M2 receptor internalization. (A) HEK 293T cells were depleted of flotillin-1 (F1), flotillin-2 (F2) or both (F1 + F2) by specific siRNAs and transfected with M2-EGFP. The cells were starved for 18 h in serum-free medium and either left untreated or stimulated with 1 mM CCh for 15 min. Cells were fixed and immunostained for flotillin-1 and flotillin-2. The nuclei were stained with DAPI (shown in blue). Scale bars: 10 μm; (B) The fraction of cells displaying M2 receptor internalization was quantified and is shown as the percent of cells showing intracellular M2 localization. At least 100 cells were counted per condition. Results are shown as the mean ± SD. Statistical analysis was performed with two-way ANOVA; (C) Equal amounts of cell lysates were separated by SDS-PAGE and immunoblotted to monitor the knockdown efficiency.

Mentions: Flotillins, especially flotillin-1, are associated with membrane trafficking processes, including endocytosis (reviewed in [26], see also [27,28]). Our recent data show that flotillin-1 is involved in mAChR signaling in keratinocytes [29], but no data exist on the possible role of flotillins in mAChR endocytosis. Since CHC knockdown did not completely prevent M2 endocytosis, we tested if flotillins might be involved. To study this, knockdown experiments were performed with siRNAs against either or both flotillins. Reduction of flotillin levels had no influence on the M2 receptor localization in starved HEK 293T cells (Figure 3A, shown for control siRNA and double-depleted cells), and even in agonist-treated cells, depletion of one or both flotillins did not impair M2 internalization (Figure 3A). The fraction of flotillin-depleted cells showing M2 endocytosis was equal to the controls (Figure 3B). The degree of flotillin depletion was monitored by Western blot (Figure 3C). Please note that flotillin-2 depletion also impairs flotillin-1 expression, due to the reduced stability of the protein. These data show that flotillins do not play a role in the uptake of the M2 receptor from the plasma membrane.


Revisiting the endocytosis of the m2 muscarinic acetylcholine receptor.

Ockenga W, Tikkanen R - Membranes (Basel) (2015)

Knockdown of flotillin-1 and flotillin-2 has no impact on M2 receptor internalization. (A) HEK 293T cells were depleted of flotillin-1 (F1), flotillin-2 (F2) or both (F1 + F2) by specific siRNAs and transfected with M2-EGFP. The cells were starved for 18 h in serum-free medium and either left untreated or stimulated with 1 mM CCh for 15 min. Cells were fixed and immunostained for flotillin-1 and flotillin-2. The nuclei were stained with DAPI (shown in blue). Scale bars: 10 μm; (B) The fraction of cells displaying M2 receptor internalization was quantified and is shown as the percent of cells showing intracellular M2 localization. At least 100 cells were counted per condition. Results are shown as the mean ± SD. Statistical analysis was performed with two-way ANOVA; (C) Equal amounts of cell lysates were separated by SDS-PAGE and immunoblotted to monitor the knockdown efficiency.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496640&req=5

membranes-05-00197-f003: Knockdown of flotillin-1 and flotillin-2 has no impact on M2 receptor internalization. (A) HEK 293T cells were depleted of flotillin-1 (F1), flotillin-2 (F2) or both (F1 + F2) by specific siRNAs and transfected with M2-EGFP. The cells were starved for 18 h in serum-free medium and either left untreated or stimulated with 1 mM CCh for 15 min. Cells were fixed and immunostained for flotillin-1 and flotillin-2. The nuclei were stained with DAPI (shown in blue). Scale bars: 10 μm; (B) The fraction of cells displaying M2 receptor internalization was quantified and is shown as the percent of cells showing intracellular M2 localization. At least 100 cells were counted per condition. Results are shown as the mean ± SD. Statistical analysis was performed with two-way ANOVA; (C) Equal amounts of cell lysates were separated by SDS-PAGE and immunoblotted to monitor the knockdown efficiency.
Mentions: Flotillins, especially flotillin-1, are associated with membrane trafficking processes, including endocytosis (reviewed in [26], see also [27,28]). Our recent data show that flotillin-1 is involved in mAChR signaling in keratinocytes [29], but no data exist on the possible role of flotillins in mAChR endocytosis. Since CHC knockdown did not completely prevent M2 endocytosis, we tested if flotillins might be involved. To study this, knockdown experiments were performed with siRNAs against either or both flotillins. Reduction of flotillin levels had no influence on the M2 receptor localization in starved HEK 293T cells (Figure 3A, shown for control siRNA and double-depleted cells), and even in agonist-treated cells, depletion of one or both flotillins did not impair M2 internalization (Figure 3A). The fraction of flotillin-depleted cells showing M2 endocytosis was equal to the controls (Figure 3B). The degree of flotillin depletion was monitored by Western blot (Figure 3C). Please note that flotillin-2 depletion also impairs flotillin-1 expression, due to the reduced stability of the protein. These data show that flotillins do not play a role in the uptake of the M2 receptor from the plasma membrane.

Bottom Line: The uptake of the M2 receptor involves the adapter proteins of the β-arrestin family and the small GTPase ADP-ribosylation factor 6.However, it has remained inconclusive if M2 endocytosis is dependent on clathrin or the large GTPase dynamin.The expression of various dominant-negative dynamin-2 mutants and the use of chemical inhibitors of dynamin function revealed that dynamin expression and membrane localization as such appear to be necessary for M2 endocytosis, whereas dynamin GTPase activity is not required for this process.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biochemistry, Medical Faculty, University of Giessen, Friedrichstrasse 24, D-35392 Giessen, Germany. Wymke.Ockenga@biochemie.med.uni-giessen.de.

ABSTRACT
The agonist-induced endocytosis of the muscarinic acetylcholine receptor M2 is different from that of the other members of the muscarinic receptor family. The uptake of the M2 receptor involves the adapter proteins of the β-arrestin family and the small GTPase ADP-ribosylation factor 6. However, it has remained inconclusive if M2 endocytosis is dependent on clathrin or the large GTPase dynamin. We here show by means of knocking down the clathrin heavy chain that M2 uptake upon agonist stimulation requires clathrin. The expression of various dominant-negative dynamin-2 mutants and the use of chemical inhibitors of dynamin function revealed that dynamin expression and membrane localization as such appear to be necessary for M2 endocytosis, whereas dynamin GTPase activity is not required for this process. Based on the data from the present and from previous studies, we propose that M2 endocytosis takes place by means of an atypical clathrin-mediated pathway that may involve a specific subset of clathrin-coated pits/vesicles.

No MeSH data available.


Related in: MedlinePlus