Transit of receptors for epidermal growth factor and transferrin through clathrin-coated pits. Analysis of the kinetics of receptor entry.
Selective enrichment of clathrin-coated membranes by anticlathrin immunoadsorption was used to examine the internalization of receptor-ligand complexes through coated pits. Using Staphylococcus aureus-anticlathrin antibody and [35S]methionine-labeled KB cells, the kinetics of association of the epidermal growth factor (EGF-R) and transferrin receptors (TF-R) with coated membranes were directly examined. The accumulation of EGF-R in coated pits at the cell surface was dependent upon EGF binding. EGF-R then passed sequentially through a compartment which did not react with anticlathrin antibody and a second clathrin-coated compartment. The EGF-R was degraded in lysosomes with a half-life of approximately 41-55 min. The tumor promoter, 4 beta-phorbol 12-myristate 13-acetate, appears to mimic the action of EGF in inducing EGF-R accumulation in coated pits at the cell surface and receptor internalization. In contrast to the results with EGF-R, the TF-R was found in clathrin-coated membranes in the presence or absence of TF, and the concentration of TF-R in clathrin-coated membranes did not significantly change with time. The method presented should be of great utility for examining the biochemical changes that occur during the receptor-mediated endocytosis and sorting of ligands and receptors.
Hanover
J A
JA
Beguinot
L
L
Willingham
M C
MC
Pastan
I H
IH
D000818:Animals; D001693:Biological Transport, Active; D002966:Clathrin; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors; D006207:Half-Life; D006801:Humans; D007163:Immunosorbent Techniques; D007700:Kinetics; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011990:Receptors, Transferrin; D013499:Surface Properties; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002966:Clathrin; D011956:Receptors, Cell Surface; D011990:Receptors, Transferrin; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D013755:Tetradecanoylphorbol Acetate