Intracellular processing of ¹²⁵I‐epidermal growth factor in rat embryo fibroblasts

Abstract

The intracellular fate of endocytosed 125I-epidermal growth factor was examined in Rat-1 fibroblasts. Cells were pulse-labeled for 5 min in 125I-EGF and chased for 3 hr with an excess of unlabeled EGF. At various times after application of the cold chase, cells were harvested and processed for isopycnic gradient centrifugation on Percoll gradients. Within the period of the 125I-EGF pulse, about 50% of the 125I activity appeared in an organelle containing peak in the gradients. By 20 min after application of the cold chase, 125I activity in the organelle peak began to decrease, and the decrease continued over the next few hours. The 125I activity which exited from its organelle-associated location appeared to be present in the cytosol and was apparently not confined within organelles. Lysosomotropic amines inhibited the egress of 125I activity from the organelle compartment. The 125I activity from both organelle and nonorganelle compartments reacted as completely as authentic 125I-EGF with anti-EGF antibodies and was similar in size to authentic 125I-EGF. Little or no intracellular low molecular weight 125I-containing compounds were detected, although they accumulated in the culture medium. Analytical isoelectric focusing revealed that the organelle-bound form of endocytosed 125I-EGF was more acidic than authentic 125I-EGF and, upon exiting from the organelle compartment, was processed to an even more acidic form. It was the second macromolecular form of processed 125I-EGF that was ultimately degraded to low molecular weight compounds which were then externalized from the cells.

Physiological functions of endosomal proteolysis

“…Peptide hormones are taken up in target cells by receptormediated endocytosis and are subsequently processed in the endocytic pathway. Four types ofprocessing have been observed: (a) EGF is partially degraded in prelysosomal compartments and then completely degraded in lysosomes [93][94][95][96][97][98][99]; (b) parathyroid hormone (PTH) is activated in macrophages by partial proteolysis in endosomes, and subsequently released to act on target cells [90,100-102]; (c) insulin and glucagon are, at least in some target cells, completely degraded in endosomes [103][104][105][106][107][108][109][110][111]; (d) some peptide hormones may be degraded in lysosomes ( Table 1). The physiological role of endosomal proteolysis of hormones is not fully understood.…”

Monoclonal antibody against epidermal growth factor receptor is internalized without stimulating receptor phosphorylation.

“…Percoll gradient fractionation was performed as described (26), except that the Percoll concentration was 35%. Cells were disrupted by resuspending in 1.0 ml of acetate triethanolamine buffer (1 mM EDTA/10 mM acetic acid/10 mM triethanolamine, pH 7.4) and lysed by pipetting the suspension 20-40 times with a P1000 Pipetman (27) 20,000 x g for 1 hr, the supernatant (250,ul) was subjected to immunoprecipitation with 2 jig of anti-EGF receptor mAb-528, 4.5 ,ug of rabbit anti-mouse antibody, and Pansorbin as described (28).…”

“…2). This B 00 0~~~õ peak is known to contain receptosomes (26,27). The density of this organelle-containing fraction was 1.04 g/ml.…”

Resolution of multiple endosomal compartments associated with the internalization of epidermal growth factor and transferrin

“…This contrasts with EGF, which is found in both E3 and E4. Furthermore, several laboratories (Magun et al, 1982;Matrisian et al, 1984;Schaudies and Savage, 1986) have described C-terminal proteolytic digestion of EGF during its transit from the cell surface to secondary lysosomes. This proteolysis results in the sequential removal of one, four, and then one aminoacyl residue.…”