Transit of receptors for epidermal growth factor and transferrin through clathrin-coated pits. Analysis of the kinetics of receptor entry.

J A Hanover,L Beguinot,M C Willingham,I H Pastan

doi:10.1016/s0021-9258(17)36349-4

Abstract

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.

Highlights

Selective enrichment of clathrin-coated membranes face prior to ligand binding; for example, the EGF’ receptor by anticlathrin immunoadsorptionwas used to exam- [13, 14] appears to behave in this fashion
Molecular ticlathrinantibodyand[36S]methionine-labeled KB approaches toward understanding the relationship between cells, the kinetics ofassociation oftheepidermal receptor structure and function have been hampered by the growth factor (EGF-R) and transferrin receptors (TF-lack of appropriate techniques for examining the various R) with coatedmembranes were directlyexamined. stages of endocytosis biochemically
Oneclue to thelocation of the sorting signals involved to the results with EGF-R,theTF-R was foundin inthis divergence has emerged from electron microscopic clathrin-coated membranes in the presencoer absence observation of peroxidase conjugates of EGF (EGFhorseradof TF, and the concentration of TF-R in clathrin-coaisthedperoxidase) and TF (TF horseradish peroxidase) during membranes did nostignificantlychange with time

Summary

Diseases and the

Selective enrichment of clathrin-coated membranes face prior to ligand binding; for example, the EGF’ receptor by anticlathrin immunoadsorptionwas used to exam- [13, 14] appears to behave in this fashion. EGF horseradish peroxidase was found method presented should be of greauttility for exam- to proceed from the cell surface through coated pits and ining the biochemical changes that occur during the endocytic vesicles into tubular structuresof the Golgi region, receptor-mediated endocytosis and sortingof ligands where it was found concentrated in the small coated pits of and receptors. We have used immunoadsorption of clathrin-coated membranes to examine ies indicate that coated pits selectively concentrate receptors directly the kinetics of association of EGFand TF with and ligands taken up by receptor-mediated endocytosis. Aliquots of the RIPA extracts wereremoved for subsequent trichloroacetic association of TF-R with clathrin-coated membranes is dif- acid precipitation to examine the polypeptides present, and the referent from that of EGF-R; the amount of TF-R associated with coated membranes was not dependent on T F binding and did not change significantly with time. Were obtained from the American Type Culture Collection and maintained in cultureas previously described [16]

RESULTS

Heavy Chain

Warmup time

DISCUSSION