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.
ASJC Scopus subject areas
- Molecular Biology