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Clathrin-mediated endocytosis in AP-2-depleted cells.

Motley A, Bright NA, Seaman MN, Robinson MS - J. Cell Biol. (2003)

Bottom Line: Receptor-mediated endocytosis of transferrin was severely inhibited in both clathrin- and AP-2-depleted cells.These results indicate that AP-2 is not essential for clathrin-coated vesicle formation at the plasma membrane, but that it is one of several endocytic adaptors required for the uptake of certain cargo proteins including the transferrin receptor.Uptake of the EGF and LDL receptors may be facilitated by alternative adaptors.

View Article: PubMed Central - PubMed

Affiliation: University of Cambridge, Department of Clinical Biochemistry, Cambridge Institute for Medical Research, Cambridge CB2 2XY, UK.

ABSTRACT
We have used RNA interference to knock down the AP-2 mu2 subunit and clathrin heavy chain to undetectable levels in HeLaM cells. Clathrin-coated pits associated with the plasma membrane were still present in the AP-2-depleted cells, but they were 12-fold less abundant than in control cells. No clathrin-coated pits or vesicles could be detected in the clathrin-depleted cells, and post-Golgi membrane compartments were swollen. Receptor-mediated endocytosis of transferrin was severely inhibited in both clathrin- and AP-2-depleted cells. Endocytosis of EGF, and of an LDL receptor chimera, were also inhibited in the clathrin-depleted cells; however, both were internalized as efficiently in the AP-2-depleted cells as in control cells. These results indicate that AP-2 is not essential for clathrin-coated vesicle formation at the plasma membrane, but that it is one of several endocytic adaptors required for the uptake of certain cargo proteins including the transferrin receptor. Uptake of the EGF and LDL receptors may be facilitated by alternative adaptors.

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Related in: MedlinePlus

Model showing interactions at the plasma membrane in control and siRNA-treated cells. In control cells, AP-2 and alternative adaptors are both recruited onto the plasma membrane, where they interact with each other and recruit clathrin through interactions mainly involving their flexible linker and appendage domains, whereas their more highly structured domains interact with phosphoinositides and with cargo. In AP-2–depleted cells, only alternative adaptors are recruited onto the plasma membrane, but these can still associate with each other (e.g., through EH domain–NPF interactions and SH3 domain–proline-rich domain interactions), and can still recruit clathrin and bring a subset of cargo proteins into the coated pit. However, without AP-2 there is less clathrin on the plasma membrane and fewer coated pits per cell, and cargo proteins that only have signals for AP-2 do not get internalized into coated vesicles. In clathrin-depleted cells, AP-2 and alternative adaptors are both recruited onto the plasma membrane, where they interact with each other and with cargo, but the membrane does not invaginate and there is no selective uptake of cargo.
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fig6: Model showing interactions at the plasma membrane in control and siRNA-treated cells. In control cells, AP-2 and alternative adaptors are both recruited onto the plasma membrane, where they interact with each other and recruit clathrin through interactions mainly involving their flexible linker and appendage domains, whereas their more highly structured domains interact with phosphoinositides and with cargo. In AP-2–depleted cells, only alternative adaptors are recruited onto the plasma membrane, but these can still associate with each other (e.g., through EH domain–NPF interactions and SH3 domain–proline-rich domain interactions), and can still recruit clathrin and bring a subset of cargo proteins into the coated pit. However, without AP-2 there is less clathrin on the plasma membrane and fewer coated pits per cell, and cargo proteins that only have signals for AP-2 do not get internalized into coated vesicles. In clathrin-depleted cells, AP-2 and alternative adaptors are both recruited onto the plasma membrane, where they interact with each other and with cargo, but the membrane does not invaginate and there is no selective uptake of cargo.

Mentions: Fig. 6 is a schematic diagram summarizing our results and how we interpret them. Normally, clathrin, AP-2, and alternative adaptors all co-assemble at the plasma membrane, bringing cargo with different types of internalization signals into the coated pit. In the absence of AP-2, alternative adaptors are still recruited onto the plasma membrane, where they interact with a subset of the cargo proteins, including the EGF and LDL receptors, and co-assemble with clathrin. These cargo proteins are still efficiently internalized, but cargo proteins like the transferrin receptor, which can only interact with AP-2, stay on the cell surface. In addition, because AP-2 is the major clathrin adaptor at the plasma membrane, there is less clathrin recruited onto the membrane and fewer (and perhaps smaller) coated pits per cell. In the absence of clathrin, both AP-2 and alternative adaptors are still recruited onto the plasma membrane, where they interact with each other and most likely with potential cargo proteins as well, but there are no morphologically recognizable coated pits, and all of the cargo proteins are internalized much more slowly.


Clathrin-mediated endocytosis in AP-2-depleted cells.

Motley A, Bright NA, Seaman MN, Robinson MS - J. Cell Biol. (2003)

Model showing interactions at the plasma membrane in control and siRNA-treated cells. In control cells, AP-2 and alternative adaptors are both recruited onto the plasma membrane, where they interact with each other and recruit clathrin through interactions mainly involving their flexible linker and appendage domains, whereas their more highly structured domains interact with phosphoinositides and with cargo. In AP-2–depleted cells, only alternative adaptors are recruited onto the plasma membrane, but these can still associate with each other (e.g., through EH domain–NPF interactions and SH3 domain–proline-rich domain interactions), and can still recruit clathrin and bring a subset of cargo proteins into the coated pit. However, without AP-2 there is less clathrin on the plasma membrane and fewer coated pits per cell, and cargo proteins that only have signals for AP-2 do not get internalized into coated vesicles. In clathrin-depleted cells, AP-2 and alternative adaptors are both recruited onto the plasma membrane, where they interact with each other and with cargo, but the membrane does not invaginate and there is no selective uptake of cargo.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172830&req=5

fig6: Model showing interactions at the plasma membrane in control and siRNA-treated cells. In control cells, AP-2 and alternative adaptors are both recruited onto the plasma membrane, where they interact with each other and recruit clathrin through interactions mainly involving their flexible linker and appendage domains, whereas their more highly structured domains interact with phosphoinositides and with cargo. In AP-2–depleted cells, only alternative adaptors are recruited onto the plasma membrane, but these can still associate with each other (e.g., through EH domain–NPF interactions and SH3 domain–proline-rich domain interactions), and can still recruit clathrin and bring a subset of cargo proteins into the coated pit. However, without AP-2 there is less clathrin on the plasma membrane and fewer coated pits per cell, and cargo proteins that only have signals for AP-2 do not get internalized into coated vesicles. In clathrin-depleted cells, AP-2 and alternative adaptors are both recruited onto the plasma membrane, where they interact with each other and with cargo, but the membrane does not invaginate and there is no selective uptake of cargo.
Mentions: Fig. 6 is a schematic diagram summarizing our results and how we interpret them. Normally, clathrin, AP-2, and alternative adaptors all co-assemble at the plasma membrane, bringing cargo with different types of internalization signals into the coated pit. In the absence of AP-2, alternative adaptors are still recruited onto the plasma membrane, where they interact with a subset of the cargo proteins, including the EGF and LDL receptors, and co-assemble with clathrin. These cargo proteins are still efficiently internalized, but cargo proteins like the transferrin receptor, which can only interact with AP-2, stay on the cell surface. In addition, because AP-2 is the major clathrin adaptor at the plasma membrane, there is less clathrin recruited onto the membrane and fewer (and perhaps smaller) coated pits per cell. In the absence of clathrin, both AP-2 and alternative adaptors are still recruited onto the plasma membrane, where they interact with each other and most likely with potential cargo proteins as well, but there are no morphologically recognizable coated pits, and all of the cargo proteins are internalized much more slowly.

Bottom Line: Receptor-mediated endocytosis of transferrin was severely inhibited in both clathrin- and AP-2-depleted cells.These results indicate that AP-2 is not essential for clathrin-coated vesicle formation at the plasma membrane, but that it is one of several endocytic adaptors required for the uptake of certain cargo proteins including the transferrin receptor.Uptake of the EGF and LDL receptors may be facilitated by alternative adaptors.

View Article: PubMed Central - PubMed

Affiliation: University of Cambridge, Department of Clinical Biochemistry, Cambridge Institute for Medical Research, Cambridge CB2 2XY, UK.

ABSTRACT
We have used RNA interference to knock down the AP-2 mu2 subunit and clathrin heavy chain to undetectable levels in HeLaM cells. Clathrin-coated pits associated with the plasma membrane were still present in the AP-2-depleted cells, but they were 12-fold less abundant than in control cells. No clathrin-coated pits or vesicles could be detected in the clathrin-depleted cells, and post-Golgi membrane compartments were swollen. Receptor-mediated endocytosis of transferrin was severely inhibited in both clathrin- and AP-2-depleted cells. Endocytosis of EGF, and of an LDL receptor chimera, were also inhibited in the clathrin-depleted cells; however, both were internalized as efficiently in the AP-2-depleted cells as in control cells. These results indicate that AP-2 is not essential for clathrin-coated vesicle formation at the plasma membrane, but that it is one of several endocytic adaptors required for the uptake of certain cargo proteins including the transferrin receptor. Uptake of the EGF and LDL receptors may be facilitated by alternative adaptors.

Show MeSH
Related in: MedlinePlus