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Biomedical subjects

D D Cunningham

Publications and source records attributed to D D Cunningham.

At least 91 records · Page 5Linked to original sources
PubMed1982

Epidermal growth factor stimulates fluid phase endocytosis in human fibroblasts through a signal generated at the cell surface.

We have investigated the stimulation of fluid phase endocytosis by epidermal growth factor (EGF) in normal human fibroblasts using 125I-labeled polyvinylpyrrolidone (125I-PVP) as a fluid phase marker. We found that EGF initially induced a threefold increase in the rate of 125I-PVP uptake. This initial burst of fluid uptake terminated within 10 min. Thereafter, the rate of fluid uptake in EGF-treated cells was approximately 40% higher than in control cells. To identify the cellular site of EGF action in stimulating fluid phase endocytosis, we examined the kinetics of the induction of this response as well as the kinetics of cell surface binding and internalization of 125I-EGF. Although there was no detectable lag between binding of EGF to the cell surface and its internalization, the kinetics of the two processes were quite different. Significantly, the kinetics of induction of 125I-PVP uptake matched the kinetics of binding of 125I-EGF to its cell surface receptors, indicating that the signal for the increase in fluid phase endocytosis is generated at the cell surface. To determine if EGF-stimulated fluid phase endocytosis was related to EGF-stimulated endocytosis of its own receptor, we compared the EGF dose dependency and time course of the two processes. Although the stimulated endocytosis of the EGF receptor was not saturable with respect to the concentration of EGF used, the stimulation of fluid phase endocytosis was half maximal at an EGF concentration of 1 ng/ml and saturated at a concentration of 5 ng/ml. Also, the stimulation of fluid phase endocytosis was sevenfold greater initially after adding EGF than after a 30-min continuous incubation with the hormone, whereas the enhanced clearance of the EGF receptor did not change during this time period. We conclude that the EGF-stimulated increase in fluid phase endocytosis is not directly coupled to EGF-stimulated endocytosis of its own receptor but instead to a separate signal generated at the cell surface.

Cells, Cultured↗
PubMed1982

Epidermal growth factor carrier protein binds to cells via a complex with released carried protein nexin.

Epidermal growth factor carrier protein (CP) is an arginine endopeptidase bound to epidermal growth factor (EGF) in vivo that processes pro-EGF to EGF and potentiates EGF action. Here, we provide a base for studying the biological functions of CP by showing that highly purified 125I-labeled CP, free of contaminating EGF, is specifically bound and internalized by normal human fibroblasts in serum-free medium. The characteristics of the binding reaction, however, were unusual and not consistent with direct interaction of CP with cell surface receptors. Subsequent experiments showed that cellular binding of 125I-labeled CP was mediated via a cell-secreted protein. We named the protein carrier protein nexin (CPN) because of its close functional similarity to protease nexin, which mediates cellular binding of thrombin or urokinase. Both CPN and protease nexin are secreted by cells, form covalent complexes with regulatory proteases in the extracellular environment, and mediate cellular binding of these proteases, apparently via a cell surface receptor for the nexin moiety of the complex. By several criteria, however, CPN and protease nexin are unique entities. This finding of a specific interaction of a growth factor carrier protein with cells suggests the possibility of additional physiological functions for these carriers in growth factor action or metabolism or both.

Animals↗
PubMed1981

Cleavage of cell surface proteins by thrombin.

This study was based on our previous findings that the mitogenic action of thrombin on cultured fibroblasts can result from interaction of thrombin with the cell surface in the absence of internalization, and that the proteolytic activity of thrombin is required for stimulation of cell division. This prompted us to look for thrombin-mediated cleavages using 2-dimensional gel electrophoresis of labeled cell surface proteins. Surface membrane components were labeled by 3 procedures: 1) proteins were labeled by lactoperoxidase-catalyzed iodination using 125I-; 2) galactose and galactosamine residues of glycoproteins were oxidized with galactose oxidase and reduced with 3H-NaBH4; and 3) glycoproteins were metabolically labeled by incubating cells with 3H-fucose. labeling with the first 2 procedures was carried out after thrombin treatment; in contrast, cells metabolically labeled with 3H-fucose were subsequently treated with thrombin to look for proteolytic cleavages. Collectively, these studies indicated that only about 5 cell surface proteins were thrombin-sensitive, consistent with the high specificity of this protease. Each of the labeling procedures revealed a thrombin-sensitive cell surface glycoprotein which was identified as fibronectin by immunoprecipitation experiments. In addition, cell surface proteins of about 140K and 55K daltons were thrombin-sensitive. However, cell surface proteins of about 45K daltons and 130K to 150K daltons were increased after thrombin treatment. These experiments were conducted on an established line of Chinese hamster lung cells with the eventual goal of studying thrombin-mediated cleavages of cell surface proteins in a large number or in cloned populations derived from this line that are either responsive or unresponsive to the mitogenic action of thrombin. This approach should permit identification of proteolytic cleavages tha are necessary for thrombin-stimulated cell division.

Animals↗
PubMed1981

Released protease-nexin regulates cellular binding, internalization, and degradation of serine proteases.

Protease-nexin (PN), a component released by normal human fibroblasts into culture medium, forms covalent linkages with thrombin (Th) and the urinary plasminogen activator urokinase, apparently with their catalytic site serines. The present studies explored the function of PN by examining the interaction of protease-PN complexes with human fibroblasts and the consequences of this interaction. Th-PN and urokinase-PN complexes bind to cells via the PN portion of the complexes. The binding is selectively inhibited by heparin. Because PN has a heparin-binding site, this indicates that protease-PN complexes might bind to a cellular heparin-like site. After binding, the complexes are internalized. By inhibiting endocytosis with phenylarsine oxide, which does not affect cellular binding of Th-PN complexes, we showed that complexes must be internalized before they are degraded. Kinetic analysis of internalization and degradation of Th-PN showed that complexes are internalized more rapidly than they dissociate from the cell surface; by 120 min of incubation at 37 degrees C most cell-bound Th-PN complexes are degraded to amino acids. The results are summarized in a model showing how PN mediates the cellular binding, internalization, and degradation of serine proteases through formation of protease-PN complexes. This series of events may be involved in the regulation of serine protease activity at the cell surface and in the extracellular environment.

Amyloid beta-Protein Precursor↗
PubMed1980

Protease-nexin: a cellular component that links thrombin and plasminogen activator and mediates their binding to cells.

This report identifies a component of normal human fibroblasts that forms a covalent linkage with thrombin and urokinase (urinary plasmingoen activator) and mediates most of the specific cellular binding of these proteases. This component, here named protease-nexin (PN), is both associated with the cell surface and released into the culture medium. In several ways PN resembles antithrombin III (AT3), a prominent inhibitor of thrombin in serum: PN links thrombin, probably via an ester bond; PN does not link thrombin blocked at its catalytic site serine; PN has a high-affinity heparin-binding site; and heparin greatly accelerates the rate of linkage between soluble PN and thrombin. Despite these similarities, PN and AT3 are distinct; they differ in size and are not immunologically cross-reactive. Whereas AT3 regulates the proteolytic activity of thrombin in serum, PN may regulates the activity of serine proteases at and near the cell surface.

Amyloid beta-Protein Precursor↗
PubMed1979

Thrombin-stimulated cell division involves proteolysis of its cell surface receptor.

A cell-surface component of molecular weight 43,000 is cleaved by thrombin on cells that divide after thrombin treatment, but is not cleaved on cells that are unresponsive to its mitogenic action. Studies with a photoreactive derivative of thrombin showed that its cell surface receptor has a molecular weight of 43,000. This indicates that thrombin must cleave its receptor to stimulate cell division.

Animals↗
PubMed1979

Direct linkage of thrombin to its cell surface receptors in different cell types.

When 125I-thrombin was incubated with foreskin fibroblasts, cervical carcinoma cells or fibrosarcoma cells of human origin, or with secondary chick embryo cells or Chinese hamster lung cells, it became directly linked to its cell surface receptors. The thrombin-receptor complex (TH-R) was derived exclusively from a pool of 125I-thrombin that had become specifically bound to the cell surface. The linkage was probably covalent, since the complex was resistant to boiling in sodium dodecyl sulfate and 2-mercaptoethanol. Raising the pH to 12 disrupted TH-R, but did not affect a similar complex between epidermal growth factor and its receptor, suggesting that the linkage of these mitogens to their receptors was different. Mild trypsin treatment removed the ability of cells to form TH-R; however, after a 24-h incubation in serum-free medium, trypsin-treated cells recovered the capacity to form TH-R, suggesting that TH-R resulted from interaction of 125I-thrombin with a cellular rather than a serum component. The mitogenic response of cells to thrombin was inversely related to the fraction of specifically bound 125I-thrombin represented by TH-R. The role of TH-R in mitogenesis may be clarified in future studies by obtaining clones of Chinese hamster lung cells that vary in their capacities to form TH-R and to respond to the mitogenic action of thrombin.

Animals↗
PubMed1978

Glucocorticoid-mediated alteration in growth factor binding and action: analysis of the binding change.

The addition of the glucocorticoid analog dexamethasone (DX) to serum-free cultures of human fibroblasts caused a twofold enhancement of the mitogenic response to epidermal growth factor (EGF), although DX by itself was not mitogenic. A basis for this effect was suggested by studies showing that DX also increased the cellular binding of 125I-EGF. DX increased the ability of the cells to bind 125I-EGF only at low physiological concentrations of this polypeptide. Thus, data from 125 I-EGF binding to cells incubated without DX produced a linear Scatchard plot, whereas the data from 125I-EGF binding to DX-treated cells led to an upwardly curvilinear Scatchard plot. Measurements of 125I-EGF association with the cell surface and cytoplasm indicated that this binding change involved an alteration of cell surface EGF receptors. The binding change appeared not to involve negatively cooperative interactions between EGF receptors, nor a change in the number of receptors. The binding alteration could be explained by a model in which DX converted 25--30% of the cell surface EGF receptors to a form having a fourfold increased affinity.

Cell Division↗
PubMed1978

Transmembrane action of thrombin initiates chick cell division.

Thrombin immobilized on polystyrene beads initiates DNA synthesis and cell division in quiescent cultures of chick embryo (CE) cells in serum-free medium. These thrombin beads also produce morphological changes in CE cells similar to those produced by soluble thrombin. The amount of acid-precipitable material released from 125I-thrombin-beads into the culture medium was 60-fold less than the amount of soluble thrombin required to produce an equivalent increase in cell number. Moreover, EM autoradiography of CE cells 10 h after 125I-thrombin-bead addition showed that there was no direct release and accumulation of radioactive material in the cytoplasm of these cells. These results demonstrate that thrombin action at the cell surface is sufficient to initiate proliferation of CE cells.

Animals↗