Morphologic characterization of the pathway of transferrin endocytosis and recycling in human KB cells.

Abstract

The pathway of transferrin uptake and recycling was investigated in KB cells to attempt to identify the organelles involved in the return of transferrin to the cell surface. Comparison was made with the pathway of internalization of epidermal growth factor (EGF), which has been shown to terminate in lysosomes. A horseradish peroxidase coIjugate of transferrin (TF-HRP) was incubated with KB cells at 40C and, at various times after warming to 370C, the location of TF-HRP was examined at the electron microscopic level. Transferrin, like EGF, was found to enter cells via coated pits and to move to the Golgi region in receptosomes (endosomes). Transferrin was located in the tubular elements of the transreticular portion of the Golgi but not in Golgi stacks. Interestingly, transferrin was not concentrated in the coated pits of the Golgi. In contrast, EGF was highly concentrated there. Transferrin was next detected in tubular elements (=600 A in width and up to 5,000 A in length) that were closely associated with microtubules and in dumbbell-shaped structures. In contrast, EGF was not detected in these structures. These results suggest that those organelles containing transferrin, but not EGF, participate in the return of receptor-bound transferrin to the cell surface.The entry of many ligands into cells by receptor-mediated endocytosis has been characterized by using both morphological and biochemical methods (1, 2). In most of these studies, ligand bound to a surface receptor can be found in clathrin-coated pits at the cell surface within seconds to minutes after binding to a surface receptor. After less than another minute, the ligand is found in intracellular endocytic vesicles termed receptosomes (or endosomes) (3-5). Receptosomes are acidic vesicles (6) that move by saltatory motion (1, 3). They have been shown to carry ligands such as epidermal growth factor (EGF) (7) and 0-galactosidase (8) from the cell surface to the cell interior, where they were delivered into the transreticular elements of the Golgi system (TRG). From the TRG these ligands and presumably many others are transferred to lysosomes, where they can be degraded (1, 2).Previous studies from our laboratory have presented data on the entry of EGF into KB cells using a covalent conjugate of EGF with horseradish peroxidase (EGF-HRP) (7, 9). After entry through coated pits the ligand was found first in receptosomes and then in the TRG. There it became concentrated in the small clathrin-coated pits of the TRG prior to its delivery to lysosomes. The suggestion was made that the role of these small coated pits of the TRG might be to segregate and concentrate those materials destined for delivery to lysosomes.The current study examines the pathway of transferrin entry and compares it with the pathway followed by EGF (7,9). Transferrin is of particular interest because after releasing its iron into the cell, it is returned intact to the extracellular medium instead of being directed to lysosomes like EGF (10-17). Thus, by comparing the intracellula...

The mode of action of methotrexate‐monoclonal antibody conjugates

“…Once the MTX-MoAb conjugates have bound their antigens/receptors -the conjugate then follows one of two different routes: either a classical receptor-mediated pathway (23) or, secondly, a vesicle containing the MTX-MoAb conjugate directly fuses with the lysosome. In the latter case the whole MTX-MoAb conjugate-antigen complex would be degraded and the antigen would be unable to return to the cell surface.…”

Molecular intactness of transferrin recycled in a myogenic chicken cell culture.

“…Iron-loaded Tf is taken up by a Tf receptor and internalized via the coated pit and receptosome (or endosome). The path of the Tf ligand is of particular interest because, after delivering its iron to a cell within an acidic nonlysosomal organelle, it returns intact to the extracellular medium instead of being directed to the lysosome as are other ligands (4,5,8,12,18,24). The molecular intactness of recycled Tf has not yet been satisfactorily demonstrated.…”

Development of novel chimeric transmembrane proteins for multimodality imaging of cancer cells

“…In all cases an SA domain is present. As seen in Figure 1A, hTrFrRp-TCA has had an internalization signal (YTRF) 36,37 and protein kinase C phosphorylation site 38 removed. In addition, a putative cathepsin G site (PKTE) 39 was changed to SSDGPGE while a basolateral signal motif (VDGDNSHVEMKLA) 36 and fatty acid acylation site 40,41 were retained.…”

“…These proteins have been extensively characterized [34][35][36][37][38][39][40][41][42][43][44][45][46][47] and are known to be expressed on cancer cells. [48][49][50] The available detailed amino acid sequence information as well as expression patterns of altered native forms; e.g., truncated and mutated, of these two receptors [37][38][39][43][44][45]51 was crucial to our considerations of which alterations to the native sequences might give the best expression alternatives (Fig. 1).…”