TY - JOUR
T1 - Receptor-mediated endocytosis in semiintact cells
AU - Smythe, E.
AU - Redelmeier, T. E.
AU - Schmid, S. L.
N1 - Funding Information:
This work was supported by grants to S. L. Schmid from the National Institutes of Health (GM42445 and CA27489) and from the Lucille P. Markey Charitable Trust. S.L.S. is a Lu¢ille P. Markey Scholar. E. Smythe was supported by a NATO/SERC Postdoctoral Fellowship.
PY - 1992/1/1
Y1 - 1992/1/1
N2 - This chapter discusses receptor-mediated endocytosis in semiintact cells. Many biologically important macromolecules are internalized into cells with the complex, multistep process of receptor-mediated endocytosis. Bound ligands and their receptors are first clustered into specialized coated pit regions of the cell surface. Cell surface receptors internalized via coated pits fall into two classes. One class, including, transferrin and low-density lipoprotein (LDL) receptors, are constitutively localized in coated pits and efficiently internalized at rates independent of bound ligand. In contrast, unoccupied receptors such as epidermal growth factor (EGF) and insulin receptors are internalized at low basal rates. These events not only require the addition of coat constituents but are accompanied by morphological changes in the coat, which progresses from an initially planar structure to a highly curved, invaginated pit. Finally, a membrane fission event occurs and the coat is sealed around the newly budded coated vesicle. Molecular dissection of these biochemically diverse events is facilitated by the use of cell-free assays that allow distinct stages to be assayed independently. In the chapter, the assay described was used to examine receptor-mediated endocytosis in A431 cells. These assays allow independent measurement of coated pit assembly, invagination, and coated vesicle budding.
AB - This chapter discusses receptor-mediated endocytosis in semiintact cells. Many biologically important macromolecules are internalized into cells with the complex, multistep process of receptor-mediated endocytosis. Bound ligands and their receptors are first clustered into specialized coated pit regions of the cell surface. Cell surface receptors internalized via coated pits fall into two classes. One class, including, transferrin and low-density lipoprotein (LDL) receptors, are constitutively localized in coated pits and efficiently internalized at rates independent of bound ligand. In contrast, unoccupied receptors such as epidermal growth factor (EGF) and insulin receptors are internalized at low basal rates. These events not only require the addition of coat constituents but are accompanied by morphological changes in the coat, which progresses from an initially planar structure to a highly curved, invaginated pit. Finally, a membrane fission event occurs and the coat is sealed around the newly budded coated vesicle. Molecular dissection of these biochemically diverse events is facilitated by the use of cell-free assays that allow distinct stages to be assayed independently. In the chapter, the assay described was used to examine receptor-mediated endocytosis in A431 cells. These assays allow independent measurement of coated pit assembly, invagination, and coated vesicle budding.
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U2 - 10.1016/0076-6879(92)19024-Z
DO - 10.1016/0076-6879(92)19024-Z
M3 - Article
C2 - 1487996
AN - SCOPUS:0027017177
SN - 0076-6879
VL - 219
SP - 223
EP - 234
JO - Methods in Enzymology
JF - Methods in Enzymology
IS - C
ER -