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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

Dynamin mutants have different effects on the agonist-mediated M2 endocytosis. (A) HEK 293T cells were transfected with M2-DsRed and the indicated dynamin-2-EGFP constructs. 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 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 localization. At least 100 cells were counted per condition. Results are shown as the mean ± SD. Statistical analysis was performed with two-way ANOVA: **p < 0.01; ***p < 0.001.
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membranes-05-00197-f004: Dynamin mutants have different effects on the agonist-mediated M2 endocytosis. (A) HEK 293T cells were transfected with M2-DsRed and the indicated dynamin-2-EGFP constructs. 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 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 localization. At least 100 cells were counted per condition. Results are shown as the mean ± SD. Statistical analysis was performed with two-way ANOVA: **p < 0.01; ***p < 0.001.

Mentions: To study the role of dynamin in M2 endocytosis, wild-type (WT) and various dominant-negative mutant forms of dynamin-2 were expressed as EGFP fusions. The dynamin-2 K44A mutant is impaired in its GTP binding capacity and, thus, exhibits lower GTP hydrolysis [30], whereas the T65A mutant is able to bind GTP, but is defective in the GTPase activity [31]. The R399A substitution leads to an impaired self-assembly and membrane localization of dynamin-2 [32]. The dynamin-2-EGFP constructs were cotransfected with M2-DsRed fusions in HEK 293T cells. The WT dynamin-2 and some of the mutants exhibited a very high level of expression and tended to form aggregates that possessed a very high fluorescence, and a corresponding signal was also detectable in the red channel for most of these aggregates (Figure 4A, left). This may indicate that the M2 receptor co-aggregated together with dynamin in these spots. However, except for these unspecific dotty signals that may also partially result from signal overflow, the M2-DsRed fusion protein was localized at the plasma membrane in starved cells. None of the expressed dynamin-2 mutants altered the M2 localization in starved HEK 293T cells. Expression of dynamin-2 WT or K44A did not prevent M2 receptor endocytosis in CCh-stimulated cells, whereas T65A produced a mild reduction in the fraction of cells exhibiting M2 endocytosis (Figure 4A, right, and 4B). However, in cells expressing the R399A mutant, M2 endocytosis was significantly impaired, and only some 20% of the cells showed M2 endocytosis, as compared to 92% of WT-expressing cells (Figure 4B).


Revisiting the endocytosis of the m2 muscarinic acetylcholine receptor.

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

Dynamin mutants have different effects on the agonist-mediated M2 endocytosis. (A) HEK 293T cells were transfected with M2-DsRed and the indicated dynamin-2-EGFP constructs. 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 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 localization. At least 100 cells were counted per condition. Results are shown as the mean ± SD. Statistical analysis was performed with two-way ANOVA: **p < 0.01; ***p < 0.001.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4496640&req=5

membranes-05-00197-f004: Dynamin mutants have different effects on the agonist-mediated M2 endocytosis. (A) HEK 293T cells were transfected with M2-DsRed and the indicated dynamin-2-EGFP constructs. 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 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 localization. At least 100 cells were counted per condition. Results are shown as the mean ± SD. Statistical analysis was performed with two-way ANOVA: **p < 0.01; ***p < 0.001.
Mentions: To study the role of dynamin in M2 endocytosis, wild-type (WT) and various dominant-negative mutant forms of dynamin-2 were expressed as EGFP fusions. The dynamin-2 K44A mutant is impaired in its GTP binding capacity and, thus, exhibits lower GTP hydrolysis [30], whereas the T65A mutant is able to bind GTP, but is defective in the GTPase activity [31]. The R399A substitution leads to an impaired self-assembly and membrane localization of dynamin-2 [32]. The dynamin-2-EGFP constructs were cotransfected with M2-DsRed fusions in HEK 293T cells. The WT dynamin-2 and some of the mutants exhibited a very high level of expression and tended to form aggregates that possessed a very high fluorescence, and a corresponding signal was also detectable in the red channel for most of these aggregates (Figure 4A, left). This may indicate that the M2 receptor co-aggregated together with dynamin in these spots. However, except for these unspecific dotty signals that may also partially result from signal overflow, the M2-DsRed fusion protein was localized at the plasma membrane in starved cells. None of the expressed dynamin-2 mutants altered the M2 localization in starved HEK 293T cells. Expression of dynamin-2 WT or K44A did not prevent M2 receptor endocytosis in CCh-stimulated cells, whereas T65A produced a mild reduction in the fraction of cells exhibiting M2 endocytosis (Figure 4A, right, and 4B). However, in cells expressing the R399A mutant, M2 endocytosis was significantly impaired, and only some 20% of the cells showed M2 endocytosis, as compared to 92% of WT-expressing cells (Figure 4B).

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