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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

MiTMAB efficiently inhibits CCh-induced M2 endocytosis. (A) HEK 293T cells were transfected with M2-EGFP, starved for 18 h in serum-free medium and pretreated with 30 μM MiTMAB. Cells were left untreated, stimulated with 1 mM CCh for 15 min or incubated with Tfn546. Cells were fixed, and 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 percentage of cells exhibiting intracellular M2. At least 100 cells were counted per condition. Results are shown as the mean ± SD. Statistical analysis was performed with two-way ANOVA: ***p < 0.001.
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membranes-05-00197-f006: MiTMAB efficiently inhibits CCh-induced M2 endocytosis. (A) HEK 293T cells were transfected with M2-EGFP, starved for 18 h in serum-free medium and pretreated with 30 μM MiTMAB. Cells were left untreated, stimulated with 1 mM CCh for 15 min or incubated with Tfn546. Cells were fixed, and 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 percentage of cells exhibiting intracellular M2. At least 100 cells were counted per condition. Results are shown as the mean ± SD. Statistical analysis was performed with two-way ANOVA: ***p < 0.001.

Mentions: In contrast to Dynasore which inhibits dynamin GTPase activity, MiTMAB prevents dynamin membrane association. Intriguingly, MiTMAB very profoundly inhibited M2 endocytosis to almost basal levels (Figure 6A,B). This degree of inhibition was very similar to that observed for the R399A dynamin-2 mutant, which is not capable of associating with membranes. Thus, our data imply that although dynamin GTPase activity appears to be largely dispensable for M2 endocytosis, dynamin as such is required and needs to be associated with membranes in order to facilitate M2 endocytosis.


Revisiting the endocytosis of the m2 muscarinic acetylcholine receptor.

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

MiTMAB efficiently inhibits CCh-induced M2 endocytosis. (A) HEK 293T cells were transfected with M2-EGFP, starved for 18 h in serum-free medium and pretreated with 30 μM MiTMAB. Cells were left untreated, stimulated with 1 mM CCh for 15 min or incubated with Tfn546. Cells were fixed, and 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 percentage of cells exhibiting intracellular M2. At least 100 cells were counted per condition. Results are shown as the mean ± SD. Statistical analysis was performed with two-way ANOVA: ***p < 0.001.
© Copyright Policy
Related In: Results  -  Collection

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

membranes-05-00197-f006: MiTMAB efficiently inhibits CCh-induced M2 endocytosis. (A) HEK 293T cells were transfected with M2-EGFP, starved for 18 h in serum-free medium and pretreated with 30 μM MiTMAB. Cells were left untreated, stimulated with 1 mM CCh for 15 min or incubated with Tfn546. Cells were fixed, and 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 percentage of cells exhibiting intracellular M2. At least 100 cells were counted per condition. Results are shown as the mean ± SD. Statistical analysis was performed with two-way ANOVA: ***p < 0.001.
Mentions: In contrast to Dynasore which inhibits dynamin GTPase activity, MiTMAB prevents dynamin membrane association. Intriguingly, MiTMAB very profoundly inhibited M2 endocytosis to almost basal levels (Figure 6A,B). This degree of inhibition was very similar to that observed for the R399A dynamin-2 mutant, which is not capable of associating with membranes. Thus, our data imply that although dynamin GTPase activity appears to be largely dispensable for M2 endocytosis, dynamin as such is required and needs to be associated with membranes in order to facilitate M2 endocytosis.

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