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

Abstract

Morphological studies have indicated divergent pathways for the endocytosis of epidermal growth factor (EGF) and transferrin (Tf). In order to obtain biochemical evidence for the pathways associated with the endocytosis of EGF and Tf, a series of Percoll density gradients were employed to separate individual cellular components. Subcellular fractionation of murine fibroblasts exposed to a 2-min pulse of either 125I-Tf or 125I-EGF results in the detection of a total of six cellular compartments related to the internalization process of these ligands. The results of kinetic analysis of the entry of EGF into five membranous fractions is consistent with a model in which ligand is transferred sequentially from the plasma membrane through three distinct prelysosomal environments prior to reaching secondary lysosomes. Each prelysosomal compartment exhibits distinct density and temporal properties in a Percoll density gradient and may represent preexisting endocytic vesicles and/or specific domains of a continuous tubular structure, vesicularized during the process of cell disruption. In addition, the observed differential migration on Percoll density gradients of Tf and EGF containing compartments indicates that the majority of cell bound Tf segregates from EGF and enters a compartment lacking EGF within 5 min of internalization.

Sorting of EGF and transferrin at the plasma membrane and by cargo-specific signaling to EEA1-enriched endosomes

“…Moreover, discrepancies in the kinetics of clearance of EGFR from the cell surface and their appearance in endosomes enriched with TfR led to speculation that vesicles containing newly internalized EGF and Tf receptors may arise as separate populations (Futter and Hopkins, 1989). Moreover, early studies involving short pulses with radiolabled EGF and Tf in fibroblasts reached the conclusion that the majority of cell bound Tf was segregated from EGF (Gorman and Poretz, 1987). Early studies using fluorescent tracers and video microscopy in live cells already revealed complexities in the endocytic pathway not anticipated by electron microscopy studies or biochemical approaches, and revealed significant differences in the pathway of internalization of Tf and EGFR (Hopkins et al, 1990).…”

The rate of internalization of different receptor–ligand complexes in alveolar macrophages is receptor-specific

“…Differences in internalization could result from the existence of ligand-specific coated pits, each of which is internalized with a characteristic rate constant. Even though recent studies suggest this possibility (Gorman & Poretz, 1987;Goldberg et al, 1987), two lines of evidence make this unlikely. First, morphological studies have demonstrated that more than one type of receptor-ligand complex can enter the same coated pit (Carpentier et al, 1982).…”

Biological effects of epidermal growth factor, with emphasis on the gastrointestinal tract and liver: An update

“…Intracellular transport of EGF via the lysosomal pathway accounts for the appearance of EGF degradation products in bile. The receptor appears to uncouple from its ligand in the acidic environment of the prelysosomal compartment and apparently proceeds with its ligand to the lysosomal compartment, where both undergo degradation (208)(209)(210). The destruction of the receptor accounts for the phenomenon known as "down-regulation" (74,75,78,79), whereby the amount of hormone taken up by a cell is self-limiting.…”