scholarly journals Kinetics of binding, endocytosis, and recycling of EGF receptor mutants

1992 ◽  
Vol 117 (1) ◽  
pp. 203-212 ◽  
Author(s):  
S Felder ◽  
J LaVin ◽  
A Ullrich ◽  
J Schlessinger
Keyword(s):  
Point Mutation ◽  
Phorbol Ester ◽  
Egf Receptor ◽  
Receptor Internalization ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Low Concentrations ◽  
Internalization Rate ◽  
Kinetics Of

This report describes analysis of factors which regulate the binding of EGF to EGF receptor, receptor internalization, and receptor recycling. Three different methods were used to inhibit high-affinity EGF binding as measured at equilibrium: treatment of cells with an active phorbol ester (PMA), binding of a mAb directed against the EGF receptor (mAb108), and truncation of most of the cytoplasmic domain of the receptor. These treatments reduced the rate at which low concentrations of EGF bound to cells, but did not affect the rate of EGF dissociation. We conclude that high-affinity EGF binding on living cells results from a difference in the apparent on rate of EGF binding. We then used these conditions and cell lines to test for the rate of EGF internalization at different concentrations of EGF. We demonstrate that internalization of the EGF receptor is stimulated roughly 50-fold at saturating concentrations of EGF, but is stimulated an additional two- to threefold at low concentrations (less than 1 nM). Four treatments reduce the rate of internalization of low concentrations of EGF to the rate seen at saturating EGF concentrations. Phorbol ester treatment and mAb108 binding to "wild type" receptor reduce this rate (and reduce high-affinity binding). Point mutation at Lys721 (kinase negative EGF receptor) and point mutation at Thr654 (removing a major site of protein kinase C phosphorylation) reduce the internalization rate, without affecting high-affinity binding. We suggest that while EGF stimulates endocytosis for all receptors, high-affinity receptors bind and are internalized more quickly than low-affinity receptors. Tyrosine kinase activity and the Thr654 region appear necessary for this response.

Heterodimerization of the epidermal-growth-factor (EGF) receptor and ErbB2 and the affinity of EGF binding are regulated by different mechanisms

2001 ◽  
Vol 356 (1) ◽  
pp. 87-96 ◽  
Author(s):  
Lene E. JOHANNESSEN ◽  
Karianne E. HAUGEN ◽  
Anne Carine ØSTVOLD ◽  
Espen STANG ◽  
Inger Helene MADSHUS
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Kinase Inhibitor ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
Receptor Dimerization ◽  
High Affinity ◽  
Epidermal Growth ◽  
Specific Kinase Inhibitor

When clathrin-dependent endocytosis is inhibited in HeLa cells by overexpression of a K44A (Lys44 → Ala) mutant of the GTPase dynamin, high-affinity binding of epidermal growth factor (EGF) to the EGF receptor (EGFR) is disrupted [Ringerike, Stang, Johannessen, Sandnes, Levy and Madshus (1998) J. Biol. Chem. 273, 16639–16642]. We now report that the effect of [K44A]dynamin on EGF binding was counteracted by incubation with the non-specific kinase inhibitor staurosporine (SSP), implying that a protein kinase is responsible for disrupted high-affinity binding of EGF upon overexpression of [K44A]dynamin. The effect of [K44A]dynamin on EGF binding was not due to altered phosphorylation of the EGFR, suggesting that the activated kinase is responsible for phosphorylation of a substrate other than EGFR. The number of EGFR molecules was increased in cells overexpressing [K44A]dynamin, while the number of proto-oncoprotein ErbB2 molecules was unaltered. EGF-induced receptor dimerization was not influenced by overexpression of [K44A]dynamin. ErbB2–EGFR heterodimer formation was found to be ligand-independent, and the number of heterodimers was not altered by overexpression of [K44A]dynamin. Neither SSP nor the phorbol ester PMA, which disrupts high-affinity EGF–EGFR interaction, had any effect on the EGFR homo- or hetero-dimerization. Furthermore, the EGF-induced tyrosine phosphorylation of ErbB2 was not affected by overexpression of [K44A]dynamin, implying that EGFR–ErbB2 dimers were fully functional. Our results strongly suggest that high-affinity binding of EGF and EGFR–ErbB2 heterodimerization are regulated by different mechanisms.

scholarly journals High-affinity Binding of Epidermal Growth Factor (EGF) to EGF Receptor Is Disrupted by Overexpression of Mutant Dynamin (K44A)

1998 ◽  
Vol 273 (27) ◽  
pp. 16639-16642 ◽  
Author(s):  
Tove Ringerike ◽  
Espen Stang ◽  
Lene E. Johannessen ◽  
Dagny Sandnes ◽  
Finn Olav Levy ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Epidermal Growth

scholarly journals Identification of an intracellular domain of the EGF receptor required for high-affinity binding of EGF

FEBS Letters ◽  
1997 ◽  
Vol 410 (2-3) ◽  
pp. 265-268 ◽  
Author(s):  
Marcel A.G Van der Heyden ◽  
Mirjam Nievers ◽  
Arie J Verkleij ◽  
Johannes Boonstra ◽  
Paul M.P Van Bergen en Henegouwen
Keyword(s):  
Egf Receptor ◽  
Affinity Binding ◽  
Intracellular Domain ◽  
High Affinity Binding ◽  
High Affinity

scholarly journals Inhibition of the apparent affinity of the epidermal growth factor receptor caused by phorbol diesters correlates with phosphorylation of threonine-654 but not other sites on the receptor

1986 ◽  
Vol 233 (2) ◽  
pp. 435-441 ◽  
Author(s):  
R J Davis ◽  
M P Czech
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Affinity Binding ◽  
Egf Receptors ◽  
A431 Cells ◽  
High Affinity Binding ◽  
Phosphorylation State ◽  
High Affinity ◽  
Epidermal Growth

Addition of 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) to A431 human epidermoid carcinoma cells causes a marked increase in the phosphorylation state of the epidermal growth factor (EGF) receptor with a concomitant inhibition of both the high-affinity binding of 125I-EGF and the receptor tyrosine kinase activity. It was found in the present studies that the diuretic drug amiloride has no effect on the action of PMA to inhibit the binding of 125I-EGF. However, amiloride was observed to inhibit markedly the effect of PMA to cause a 3-fold increase in the phosphorylation state of the EGF receptors. In the presence of PMA and amiloride, the increase in the phosphorylation state of the EGF receptors was found to be only 1.2-fold over controls. Analysis of the EGF receptor phosphorylation sites by phosphopeptide mapping by reverse-phase h.p.l.c. demonstrated that PMA increases the phosphorylation state of the EGF receptor at many sites. One of these sites has been identified as a C-kinase substrate, threonine-654. In the presence of amiloride, PMA causes phosphorylation of threonine-654 to the same stoichiometry as that observed in the absence of amiloride. However, the marked increase in the phosphorylation state of the EGF receptor at other sites caused by PMA is abolished in the presence of amiloride. We conclude that the extensive phosphorylation of the EGF receptor at several sites caused by the addition of PMA to A431 cells is not required for the action of PMA to inhibit the high-affinity binding of 125I-EGF. The results indicate that the phosphorylation state of threonine-654 may play a role in this process.

scholarly journals Endocytosis of sulphated proteoglycans by cultured skin fibroblasts

1978 ◽  
Vol 176 (3) ◽  
pp. 671-676 ◽  
Author(s):  
R Prinz ◽  
J Schwermann ◽  
E Buddecke ◽  
K von Figura
Keyword(s):  
Cell Surface ◽  
Binding Sites ◽  
Skin Fibroblasts ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
Surface Receptors ◽  
High Affinity ◽  
Cultured Skin ◽  
Kinetics Of ◽  
Adsorptive Endocytosis

1. Human skin fibroblasts internalize homologous sulphated proteoglycans by adsorptive endocytosis. Endocytosis rate is half maximal when the concentration of the proteoglycans is 0.1 nM. At saturation, a single fibroblast may endocytose up to 8 × 10(6) proteoglycan molecules/h. 2. The kinetics of prote;glycan binding to the cell surface suggest the presence of 6 × 10(5) high-affinity binding sites per cell. The bulk of sulphated proteoglycans associates to low-affinity binding sites on the cell surface. 3. Glycosaminoglycans and other anionic macromolecules inhibit endocytosis of sulphated proteoglycans non-competitively. The lack of interaction of glycosaminoglycans with the cell-surface receptors for sulphated proteoglycans suggests that the protein core of proteoglycans is essential for binding to the cell surface. 4. The effects of trypsin, cell density, serum concentration and medium pH on endocytosis and degradation of endocytosed sulphated proteoglycans is described. 5. A comparison of the number of the high-affinity binding sites and the number of molecules endocytosed with respect to time suggests a recycling of the proteoglycan receptors between the cell surface and the endocytotic vesicles and/or the lysosomes.

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