Recycling of 5′-nucleotidase in a rat hepatoma cell line.

Abstract

Intracellular movement of cell surface 5′‐nucleotidase was studied in H4S cells, a rat hepatoma cell line. Surface labelled cells were incubated for various periods at 37 degrees C and treated with neuraminidase at 0 degrees C. Removal of sialic acid residues from glycoproteins results in a change of their isoelectric points. Analysis with isoelectric focusing was then used to distinguish between cell surface and intracellular 5′‐nucleotidase. Incubation of 125I‐surface‐labelled cells at 37 degrees C resulted in a gradual decrease of labelled 5′‐nucleotidase at the plasma membrane until, at 60 to 90 min, a steady state was reached with 52% of the label on the cell surface and 48% intracellular. Pretreatment of the cells with the weak base primaquine had no influence on this distribution while at the same time uptake of iron via the transferrin receptor was inhibited. Using immunoelectron microscopy 5′‐nucleotidase was found on the cell surface, in multivesicular endosomes and the Golgi complex. Preincubation of the cells in the presence of cycloheximide caused a reduction of labelling in the Golgi complex, whereas the label in the other compartments was retained. These results lead to the conclusion that 5′‐nucleotidase does not recycle through the Golgi complex and that in contrast to the transferrin receptor the recycling of 5′‐nucleotidase is not inhibited by primaquine.

Inhibition of CD73 stimulates the migration and invasion of B16F10 melanoma cells in vitro, but results in impaired angiogenesis and reduced melanoma growth in vivo

“…A significant amount of CD73 was detected on the B16F10 cell surface by immunofluorescence; however, its main localization pattern appeared to be intracellular. This is in agreement with an earlier hypothesis stating that an extensive intracellular distribution of CD73 is present in a membrane-bound pool (i.e., lysosomes, Golgi apparatus and transcytotic vesicles) and that CD73 undergoes continual exchange between the plasmatic and internal membranes (1,32,33). We encountered a problem detecting CD73 on the surface of non-fixed B16F10 and HEK293T cells by flow cytometry (although the intracellular antigen was apparent in the fixed cells), but this might be due to ecto-domain shedding after binding of the anti-CD73 antibody.…”

A novel iron uptake mechanism mediated by GPI-anchored human p97.

“…Considering the observed difference between membrane-bound Fe for cells with and without p97 and that only 45% of p97 is removed by the pronase treatment, it appears that one molecule of p97 is able to bind one atom of Fe, which supports the conclusions of other studies (Baker et al, 1987). Previous studies have demonstrated that GPI-anchored proteins can be internalized and recycled back to the cell surface (van den Bosch et al, 1988;Tausk et al, 1989;Lemansky et al, 1990;Keller et al, 1992). The binding of Fe to p97 and the internalization of Fe is consistent with a GPI-anchored protein being involved in the binding and internalization of a ligand.…”

Sequestration of GPI-Anchored Proteins in Caveolae Triggered by Cross-Linking

“…A similar analysis (average values from five fields) of the coclustering of Thy-1 and folate receptors on G3G2 cells showed that -35% of the folate receptor clusters contained detectable Thy-1 (random colocalization value of 12%) and -55% of Thy-1 clusters contained detectable folate receptor (random colocalization value of -10%). Thus, although GPI-anchored protein monomers Im l l o o f f i j o , now that the major pathway of internalization of the folate receptor is by way of bulk membrane endocytosis (12, 13) mediated by clathrin-dependent and independent pathways (14), similar to the pathway of other GPI-anchored proteins (15,16). Previous reports that GPI-anchored proteins are excluded from coated pits (16,17) may be explained by the ability of GPI-anchored proteins to redistribute to caveolae artifactually during the process of immunolocalization, thereby apparently depleting coated pits of GPI-anchored proteins.…”