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Kinetics of binding, endocytosis, and recycling of EGF receptor mutants
1Department of Molecular Biology, Rhone-Poulenc Rorer, Inc., King of Prussia, Pennsylvania 19406.
Abstract:
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.
Insights
High-affinity epidermal growth factor (EGF) binding to its receptor results from a faster binding rate. This binding influences EGF receptor internalization, with specific mutations affecting this process.
Area of Science:
- Cell biology
- Molecular signaling
- Biochemistry
Background:
- Epidermal Growth Factor (EGF) receptor signaling is crucial for cell growth and differentiation.
- Understanding the regulation of EGF receptor binding and internalization is key to deciphering cellular responses.
Purpose of the Study:
- To investigate factors regulating EGF binding affinity and receptor internalization.
- To elucidate the role of specific receptor domains and signaling pathways in EGF receptor dynamics.
Main Methods:
- Utilized three distinct methods to inhibit high-affinity EGF binding: phorbol ester treatment (PMA), monoclonal antibody (mAb108) binding, and cytoplasmic domain truncation.
- Assessed EGF binding kinetics (on-rate and off-rate) and receptor internalization rates at varying EGF concentrations.
- Employed site-directed mutagenesis (Lys721 and Thr654) to examine the impact of kinase activity and phosphorylation on receptor function.
Main Results:
- High-affinity EGF binding is primarily determined by the apparent on-rate, not the dissociation rate.
- EGF receptor internalization is significantly stimulated by EGF, with a notable increase at low EGF concentrations.
- Phorbol ester, mAb108, Lys721 mutation, and Thr654 mutation differentially affect EGF binding affinity and/or internalization rates.
Conclusions:
- High-affinity EGF receptors bind and are internalized more rapidly than low-affinity receptors.
- Tyrosine kinase activity and the Thr654 phosphorylation site are essential for the enhanced internalization of high-affinity EGF-bound receptors.