No abstract

Abstract. Acidification of the cytosol of a number of different cell lines strongly reduced the endocytic uptake of transferrin and epidermal growth factor. The number of transferrin binding sites at the cell surface was increased in acidified cells. Electron microscopic studies showed that the number of coated pits at the cell surface was not reduced in cells with acidified cytosol. Experiments with transferrin-horseradish peroxidase conjugates and a monoclonal anti-transferrin receptor antibody demonstrated that transfen'in receptors were present in ,x,75 % of the coated pits both in control cells and in cells with acidified cytosol. The data therefore indicate that the reason for the reduced endoeytic uptake of transferrin at internal pH <6.5 is an inhibition of the pinching off of coated vesicles. In contrast, acidification of the cytosol had only little effect on the uptake of ricin and the fluid phase marker lucifer yellow. Ricin endocytosed by cells with acidified cytosol exhibited full toxic effect on the cells. Although the pathway of this uptake in acidified cells remains uncertain, some coated pits may still be involved. However, the data are also consistent with the possibility that an alternative endocytic pathway involving smooth (uncoated) pits exists.number of different transport proteins, hormones, growth factors, toxins, and viruses enter cells by receptor-mediated endocytosis. Coated pits and coated vesicles are generally assumed to be involved in this process (for reviews, see references 33, 38, and 59). Upon entry, the ligands are transferred to endosomes where the low pH is essential for dissociation of ligands from their receptors, for release of Fe 3+ from transferrin, and for entry into the cytosol of viruses and certain toxins.In studies of endocytosis and its role in the mechanism of action of many ligands, it is a problem that no specific inhibitor of endocytosis is available. Although treatments with metabolic inhibitors and low temperature inhibit endocytosis, these conditions inhibit most other physiological processes in the cells as well. Recently, removal of cytoplasmic potassium was found to inhibit endocytosis of low density lipoprotein, epidermal growth factor (EGF),~ and transferrin (29,31,37). However, such treatment does not efficiently inhibit the formation of coated vesicles in all cell types (37, 44). Furthermore, K + depletion induces cell shrinkage and inhibits anion transport (32). Alternative methods to inhibit endocytosis from coated pits are therefore warranted.In the course of our studies of the mechanism of action of diphtheria toxin, ricin, and related toxic proteins with intracellular sites of action, we noted that acidification of the cytosol inhibited endocytic uptake of different ligands to different extents (46). Here we report that acidification of the cytosol efficiently blocks endocytosis of transferrin and EGE whereas the uptake of the toxic plant protein ricin and of the membrane impermeant fluorescent dye, lucifer yellow, is only slightly reduced. Mat...

At neutral pH, NH4C1 and chloroquine protected cells against diphtheria toxin . A brief exposure of the cells to low pH (4.5-5 .5) at 37°C completely abolished this protection . When, to cells preincubated with diphtheria toxin and NH 4C1, neutralizing amounts of anti-diphtheria toxin were added before the pH was lowered, the toxic effect was considerably reduced, but it was not completely abolished . A much stronger toxic effect was seen when antibodies were added immediately after incubation at low pH .Upon a short incubation with diphtheria toxin at low pH, the rate of protein synthesis in the cells decreased much faster than when the normal pH was maintained . The data suggest that, at low pH, diphtheria toxin (or its A fragment) penetrates directly through the surface membrane of the cell . The possibility is discussed that, when the medium has a neutral pH, the entry of diphtheria toxin involves adsorptive endocytosis and reduction of the pH in the vesicles possibly by fusion with lysosomes . Low pH did not facilitate the entry of the closely related toxins abrin, ricin, and modeccin .

Translocation of ricin A chain to the cytosol has been proposed to take place from the endoplasmic reticulum (ER), but attempts to visualize ricin in this organelle have failed. Here we modified ricin A chain to contain a tyrosine sulfation site alone or in combination with N-glycosylation sites. When reconstituted with ricin B chain and incubated with cells in the presence of Na 2 35 SO 4 , the modified A chains were labeled. The labeling was prevented by brefeldin A and ilimaquinone, and it appears to take place in the Golgi apparatus. This method allows selective labeling of ricin molecules that have already been transported retrograde to this organelle. A chain containing C-terminal N-glycosylation sites became core glycosylated, indicating retrograde transport to the ER. In part of the toxin molecules, the A chain was released from the B chain and translocated to the cytosol. The finding that glycosylated A chain was present in the cytosol indicates that translocation takes place after transport of the toxin to the ER.

No abstract

No abstract

No abstract

The fibroblast growth factor receptors (FGFR) play essential roles both during development and in the adult. Upon ligand binding, FGFRs induce intracellular signaling networks that tightly regulate key biological processes, such as cell proliferation, survival, migration, and differentiation. Deregulation of FGFR signaling can thus alter tissue homeostasis and has been associated with several developmental syndromes as well as with many types of cancer. In human cancer, FGFRs have been found to be deregulated by multiple mechanisms, including aberrant expression, mutations, chromosomal rearrangements, and amplifications. In this review, we will give an overview of the main FGFR alterations described in human cancer to date and discuss their contribution to cancer progression.