Cortical actin organization is required for internalization of bombesin/GRP and EGF receptors, and ERK activation in Swiss 3T3 cells

The role of actin organization in occupancy-induced receptor internalization remains poorly defined. Here we report that treatment of mouse Swiss 3T3 cells with latrunculin A, a potent inhibitor of actin polymerization (including cortical actin), inhibited the internalization of the endogenous bombesin/gastrin-releasing peptide (GRP) receptor, as judged by uptake of 125I-labeled GRP or fluorescent Cy3-labeled bombesin. In contrast, cells pretreated with cytochalasin D showed minimal inhibition of bombesin/GRP receptor internalization. Similarly, pretreatment of Swiss 3T3 cells with the potent Rho-kinase inhibitor HA-1077, at concentrations (10–20 μM) that abrogated bombesin-mediated stress fiber formation, did not significantly alter receptor-mediated internalization of125I-GRP. These results indicate that bombesin/GRP receptor internalization depends on latrunculin A-sensitive cortical actin rather than on rapidly turning over actin stress fibers that are disrupted by either cytochalasin D or HA-1077. The rates and total levels of internalization of the endogenously expressed endothelin A receptor and epidermal growth factor receptor were also markedly reduced by latrunculin A in Swiss 3T3 cells. The potency of latrunculin A for inhibiting G protein-coupled receptor endocytosis was comparable to that for reducing internalization of the epidermal growth factor tyrosine kinase receptor. We conclude that cortical actin structures, disrupted by latrunculin A, are necessary for occupancy-induced receptor internalization in animal cells.

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EGF receptor function is required in late G1 for cell cycle progression induced by bombesin and bradykinin

AJP Cell Physiology ◽

10.1152/ajpcell.2001.281.3.c886 ◽

2001 ◽

Vol 281 (3) ◽

pp. C886-C898 ◽

Cited By ~ 42

Author(s):

Chintda Santiskulvong ◽

James Sinnett-Smith ◽

Enrique Rozengurt

Keyword(s):

Cell Cycle ◽

Tyrosine Kinase ◽

Dna Synthesis ◽

Kinase Inhibitors ◽

Egf Receptor ◽

3T3 Cells ◽

Erk Activation ◽

Egfr Tyrosine Kinase ◽

Swiss 3T3

We examined the role of epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase activation in G protein-coupled receptor (GPCR) agonist-induced mitogenesis in Swiss 3T3 and Rat-1 cells. Addition of EGFR tyrosine kinase inhibitors (e.g., tyrphostin AG-1478) abrogated bombesin-induced extracellular signal-regulated kinase (ERK) activation in Rat-1 cells but not in Swiss 3T3 cells, indicating the importance of cell context in determining the role of EGFR in ERK activation. In striking contrast, treatment with tyrphostin AG-1478 markedly (∼70%) inhibited DNA synthesis induced by bombesin in both Swiss 3T3 and Rat-1 cells. Similar inhibition of bombesin-induced DNA synthesis in Swiss 3T3 cells was obtained using four structurally different inhibitors of EGFR tyrosine kinase. Furthermore, kinetic analysis indicates that EGFR function is necessary for bombesin-induced mitogenesis in mid-late G1 in both Swiss 3T3 and Rat-1 cells. Our results indicate that EGFR kinase activity is necessary in mid-late G1 for promoting the accumulation of cyclins D1 and E and implicate EGFR function in the coupling of GPCR signaling to the activation of the cell cycle.

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Inhibition of the binding of 125I-labelled epidermal growth factor to mouse cells by a mitogen in goat mammary secretions

Biochemical Journal ◽

10.1042/bj2120465 ◽

1983 ◽

Vol 212 (2) ◽

pp. 465-472 ◽

Cited By ~ 23

Author(s):

K D Brown ◽

D M Blakeley

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Egf Receptor ◽

Competitive Inhibitor ◽

Inhibitory Effect ◽

Temperature Sensitive ◽

Egf Receptors ◽

3T3 Cells ◽

Epidermal Growth ◽

Swiss 3T3

Pre-colostrum and colostrum from goats cause a marked inhibition of the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 cells. The ability of these secretions to inhibit 125I-EGF binding is closely correlated with the ability to stimulate DNA synthesis in quiescent 3T3 cell cultures, suggesting that goat mammary secretions may contain an EGF-related mitogen. However, the material in colostrum which inhibits 125I-EGF binding to Swiss 3T3 cells is a basic protein with Mr greater than 20000 and is thus quite different from mouse and human EGF. Furthermore, the colostral-mediated inhibition of 125I-EGF binding, although rapid and apparently competitive, differs from the inhibition of binding induced by native, unlabelled EGF. Thus, the inhibitory effect of colostrum is markedly decreased when the assay temperature is shifted from 37 degrees C to 4 degrees C whereas unlabelled EGF is an effective competitive inhibitor at both 37 degrees C and 4 degrees C. Incubation of cells with EGF causes a reduction in cell surface EGF receptors whereas exposure to colostrum does not induce down-regulation of the EGF receptor. Our results suggest that the colostral factor does not bind directly to EGF receptors but inhibits 125I-EGF binding by an indirect mechanism which involves a temperature-sensitive step.

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Inhibition of epidermal growth factor binding to Swiss 3T3 cells by human platelet release-products

Bioscience Reports ◽

10.1007/bf01172876 ◽

1983 ◽

Vol 3 (7) ◽

pp. 659-666 ◽

Cited By ~ 4

Author(s):

Kenneth D. Brown ◽

Diane M. Blakeley ◽

Moira MacDonald

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

Human Platelet ◽

Platelet Derived Growth Factor ◽

Egf Receptors ◽

3T3 Cells ◽

Temperature Dependent ◽

Cellular Receptors ◽

Epidermal Growth ◽

Swiss 3T3

Human platelet ionophore release-products (IRP) inhibit the binding of 125I-labelled epidermal growth factor (125I-EGF) to its receptors on Swiss 3T3 cells. The inhibition appears to be caused by platelet-derived growth factor (PDGF) in the IRP and results from a decrease in the apparent affinity of cellular receptors for 125I-EGF. However, our results indicate that PDGF does not bind directly to EGF receptors, since (1) PDGF does not down-regulate EGF receptors; (2) the PDGF-mediated inhibition of 125I-EGF binding is temperature-dependent; (3) cells which possess EGF receptors but lack PDGF receptors do not exhibit a PDGF-mediated inhibition of 125I-EGF binding.

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