Visualization of the plasmin receptor on sections of human mammary carcinoma cells.

In previous studies we observed the presence of an antigen reacting with anti-plasminogen serum on tumor cells found in sections of human colorectal and mammary carcinomas, using immunofluorescence. This antigen could be plasminogen, or plasmin or both. These results led us to surmise that carcinoma cells, and not normal epithelial cells, had a receptor for plasmin and plasminogen. We demonstrated the existence of such a receptor on cells of established tumor cell lines of colonic and mammary origin. We observed that binding of plasmin(ogen) to its receptor was inhibited by lysin and its analogs. We undertook to characterize the plasmin receptor on sections of human mammary carcinomas (18 series of sections, originating from 15 infiltrating ductal carcinomas). In our immunofluorescence studies, we observed that the fluorescence induced by anti-plasminogen serum was suppressed by pre-treatment of the sections with tranexamic acid, a lysin analog. Furthermore, when parallel sections were incubated with tranexamic acid, then with solutions of plasminogen, tumor cells were made fluorescent again. Another important finding was the strong increase of labelling observed when sections were incubated with plasminogen or plasmin before reaction with anti-plasminogen serum. This labelling disappeared when tranexamic was added to plasminogen. As a whole, these results confirm the existence of plasmin receptors on tumor cells in mammary carcinoma sections. They also show that these receptors are not saturated in vivo and may bind, in vitro, additional amounts of ligand.

Plasminogen receptors on rat colon carcinoma cells.

Cells from rat carcinoma cell lines PROb (giving progressive tumours) and REGb (giving regressive tumours) have cell surface receptors which bind specifically rat plasminogen and plasmin. Affinity for Pg was found to be higher in PROb (Kd = 10(-7) M) than in REGb cells (Kd = 5.10(-7) M) but with a concomitant decrease in the number of binding sites, 0.9 x 10(6)/cell (range from 0.6 to 1.2 x 10(6)) in PROb vs 3.6 x 10(6)/cell (range 1.2 to 6 x 10(6)) in REGb cells. The number and the affinity of binding sites varied in an opposite way in PROb and REGb cells. The difference in affinity parameters was unrelated to the degree of invasiveness of tumour cells in syngenetic rats. Bound plasmin retained its enzymatic activity, which indicates that its binding does not involve the catalytic active site. In cell solubilisates plasminogen receptor appeared as one major band situated in the area of 50-60 kDa.

Visualization of the plasmin receptor on carcinoma cells.

In order to visualize the receptor for plasmin and plasminogen present on human carcinoma cells, SW1116 and MCF7-MF cells were incubated with biotinylated plasminogen or plasmin and fluoresceinated streptavidin, and counterstained with propidium iodide. It was first demonstrated that biotinylation did not alter the binding properties of plasminogen and plasmin, provided that the biotinylation rate was around 2. Specific staining of tumor cells was obtained using these reagents. Images with green fluorescence were clearly visible as grains or contours at the surface of tumor cells. The localization of fluorescent grains was analyzed more precisely using confocal microscopy. The percentage of stained cells varied from one experiment to another between 10 and 60%. In no experiment were all the cells observed to be positive. Mitotic cells were more frequently stained than non-mitotic cells, suggesting a relationship between the presence of plasmin receptors and cell proliferation. This was confirmed by the use of Ki67 monoclonal antibody (MAb), as B-Pg-binding tumor cells generally had their nucleus stained by this antibody. Other images indicated staining of the extracellular matrix. Finally, 2 rat tumor-cell sub-lines of colonic origin (DHD K12 Pro-b and Reg-b) were shown to bind human biotinylated plasminogen, confirming the strong interspecies reactivity of plasmin receptors.

A receptor for plasminogen and plasmin on human and rat carcinoma cells. Evidence for strong interspecific reactivity.

We have previously demonstrated the existence of a receptor for plasmin and plasminogen on carcinoma cells, either from human or rat origin. We show here that these receptors have a strong interspecific reactivity. This conclusion is based on binding assay of 125I labelled plasminogens and fluorescence experiments using biotinylated human plasminogen. In all these experiments cells from one species could bind plasminogen from the other species and interspecific inhibition was observed as well.

Clathrin beta-light chain of rat liver coated vesicles is phosphorylated in vitro and in vivo.

Clathrin beta-light chain of rat liver coated vesicles is phosphorylated in vitro in the presence of poly(L-lysine) by an endogenous protein kinase which appears to be similar to casein kinase II. Clathrin beta-light chain is also phosphorylated in vivo. After injection of [32P]phosphate into rats and preparation of purified coated vesicles in the presence of phosphatase inhibitors, electrophoretic analysis showed the presence of several labeled polypeptides including clathrin beta-light chain. A polypeptide of 50 kDa, which may correspond to the major polypeptide phosphorylated in vitro of coated vesicles, is also labeled in vivo.

Epidermal growth factor stimulated protein kinase shows similar activity in liver of senescent and adult mice.

It was recently reported [(1983) Nature 306, 617-620] that tyrosine protein kinase activity associated with EGF receptor was absent from senescent human cultured fibroblasts, which are known to have the same number of receptors as young human cultured fibroblasts. We have measured in both adult and senescent C57 black mice the number of EGF receptors, the activity of their associated tyrosine kinase and the activity of the protein phosphatase which dephosphorylates the EGF receptor. We found our results in both groups of animals to be similar which indicate that the observations made in cultured fibroblasts cannot be generalized to all mammalian tissues.

Characterization of the epidermal-growth-factor-dependent phosphorylation system from normal mouse-liver sinusoidal plasma membranes.

Blood sinusoidal plasma membrane subfractions were isolated from normal mouse liver in the presence of the proteinase inhibitors PhMeSO2F and iodoacetamide. They were purified from smooth microsomal and Golgi vesicle contaminants. The phosphorylation reaction was studied at 33 degrees C, in the presence of 2 mM MnCl2. Addition of epidermal growth factor (EGF) to the preparations stimulated 32P incorporation from [gamma-32P]ATP or [gamma-32P]GTP essentially into one 170 000 Mr protein. Some incorporation was observed in a minor 120 000-Mr component which appears to be a degradation product of the 170 000-Mr component. No EGF-dependent phosphorylation of other membrane proteins or various exogenous proteins could be detected in vitro. The dephosphorylation of the 170 000-Mr component was observed after 4 min of incubation at 33 degrees C. This dephosphorylation reaction was inhibited by addition of 5 mM p-nitrophenyl phosphate but not by addition of micromolar Zn2+, Be2+ or orthovanadate. The 170 000-Mr protein specifically bound 125I-labeled EGF and thus appeared to be the hepatic EGF receptor. The EGF stimulatable kinase activity considerably enhances incorporation of 32P into tyrosine residues of the 170 000-Mr EGF receptor at 33 degrees C. Tryptic peptide maps of the 32P-labeled 170 000-Mr protein revealed a multiplicity of phosphorylated sites. Seven 32P-labeled phosphopeptides were observed after EGF stimulation, three of them being largely prominent. Tryptic peptide maps of the 170 000-Mr protein after it was covalently linked to 125I-labeled EGF showed only one 125I-labeled peptide, the migration of which appeared different from that of 32P-labeled phosphopeptides. These findings were confirmed by V8 protease unidimensional peptide mapping of the 170 000-Mr protein, labeled with 32P or 125I-EGF.

A membrane-bound protein kinase from mouse liver stimulated by iron.

At microM levels, iron stimulates strongly the in vitro phosphorylation of a membrane protein from mouse liver. This phosphorylation also occurs in the absence of other added divalent cations but to a lower degree. In SDS gels the phosphorylated protein has app. Mr 250 000 in the absence of mercaptoethanol and Mr 130 000 in its presence. It is phosphorylated on threonine residues.