Dansylcadaverine inhibits internalization of 125I-epidermal growth factor in BALB 3T3 cells.

H.T Haigler,F.R Maxfield,M.C Willingham,I Pastan

doi:10.1016/s0021-9258(19)86019-2

Abstract

The binding and internalization of 125I-epidermal growth factor (125I-EGF) was studied in cultures of BALB 3T3 cells using a novel method that involved removal of cell-surface hormone by treatment with acetic acid under conditions that did not remove internalized hormone. In control cultures, 125I-EGF initially bound to its receptor on the plasma membrane and then was rapidly internalized. After 30 min, only 15% of the cell-bound hormone remained on the surface. In contrast, cultures treated with dansylcadaverine retained 82% of the cell-bound hormone on the cell surface. We propose that dansylcadaverine inhibits EGF internalization by preventing it from clustering in clathrin-coated pits.

Highlights

MATERIALS ANDMETHODSThe bindingandinternalizationof'251-epidermal growthfactor ("%EGF) wasstudied in cultures of
Mouse EGF was a gdt of Stanley Cohen and was iodinated (1.24 X IO5 cpm/ng) by published procedures [11].Dansylcadaverine was BALB 3T3 cells using anovelmethodthatinvolved obtained from Fulka (Switzerland)
Acetic Acid Removal of Cell Surface EGF-Confluent monolayers of BALB 3T3 cells wereincubated with lZ5I-EGF at 4°C

Summary

MATERIALS ANDMETHODS

The bindingandinternalizationof'251-epidermal growthfactor ("%EGF) wasstudied in cultures of. The internalization of epidermal growth factor, insulin, az- associated radioactivity was removed by incubating for 1h at 37°C macroglobulin, and low density lipoprotein by cultured cells with 1N NaOH. The radioactivity removed by acid and NaOH was occurs by the process of adsorptive endocytosis [1,2,3,4] These ligands bind to unoccupied receptors on the cell surface and the receptor-ligand complexes cluster in pits coated on their cytoplasmic surface with the protein clathrin [5,6,7]. The fluorescent method is very sensitive and allows one to observe the general location of hormone associated with a single cell, it does not yet allow one to quantify the amount of ligand that is bound to a cell or determine the amounts on the cell exterior and cell interior

RESULTS

Inhibition of Epidermal GIrowth Factor Internalization

The following experiment was performed to determine the

DISCUSSION