Sorting of the Receptor
Response to questions
Q. A Describe the main pathway turned ‘on’ by the EGFR to regulate cell growth
MAPK/ERK pathway is the main pathway, which is turned on by EGFR in order to regulate cell growth. This path comprises of a chain of proteins within the cell, which communicates a signal from the cell receptor surface to the DNA within the cell nucleus. When a molecule binds to the receptor at the cell surface, a signal is induced and ends when the DNA expresses a protein, which in turn produces some changes within the cell. One of the common changes are cell division. MAPK is a mitogen activated protein kinases, which was origically referred to as ERK, extracellular signal regulated kinases. It communicates signals by adding phosphate groups to adjacent proteins, which acts as an ‘ON’ or ‘OFF’ switch. The switch is referred to as the ‘Ras-Raf-MEK-ERK pathway’. One of the proteins in the pathway maybe mutated, which implies that the protein will be stuck in an ON or OFF position. This is a common step in the development of many cancers. Afterwards, the drugs that are used to reverse the ‘ON’, or ‘OFF’, switch are being investigated as probable treatments.
Q.B. During which phase of the cell cycle is EGFR activity likely to be required? Why?
EGFR is more likely required during the recycle phase of the cell cyclebased on experimental results and the current knowledge about the role of EGFR ligands in the pathogenesis of cancer, it is clear that the oncogenic potential of the various EGFRligands in part depends on their ability to induce receptorrecycling rather than degradation. Due to this EGFR is more likely to be of greater benefit at the recycle phase as opposed to any other phase of the cell cycle. During the internalization and recycling
Q.C. The authors explain that TGFalpha is a more potent mitogen then EGF. Why? Describe the specific events that occur on/ to the EGFR following binding of the two different ligands.
Following binding of the two different ligands, EGFR is internalized and trafficked to endosomes. To determine the number of unoccupied EGFRs following ligand-binding, cells were subsequently incubated with 125I-EGF for 30 min, washed, hydrolyzed in 0.5 M KOH, and the samples counted in a gamma-counter. Maximal 125I-EGF binding was set to 0% and no 125I-EGF binding to 100%.
D. How do they examine EGFR internalization from the plasma membrane?
Q.E. How did they examine EGFR localization?
For EGFR localization to early endosomes following ligand stimulation, HEp2 cells were incubated on ice with 10 nM (EGF, TGF-α, HB-EGF, and BTC) or 100 nM (AR and EPI) of ligand, washed, and incubated at 37◦C for different periods. Cells were fixedand labeled for EGFR and the early endosome marker EEA1.
For EGFR localization to lysosomes following ligand stimulation, HEp2 cells were incubated on ice with 10 nM (EGF,TGF-α, HB-EGF, and BTC) or 100 nM (AR and EPI) of ligand, washed, and incubated at 37◦C for 60 min. Cells were fixed and labeled for EGFR and the lysosomal marker Lamp1. In the case of TGF-α andEPI, relatively little EGFR is found inside the cell, whereas EGFR is distinct at the plasma membrane (arrow heads). HEp2 cells were incubatedon ice with 10 nM BTC, washed, and incubated at 37◦C in the presence of 500 nM bafilomycin A1 for 120 min. Cells were fixed andlabelled for EGFR and Lamp1. Note that bafilomycin A1 induces an up-concentration of EGFR in lysosomes following BTC stimulation.
Q.F. They used two assays for EGFR degradation; an ELISA and a pulse/chase followed by an EGFR immunoprecipitation. Explain how each assay worked, and contrast them to each other. Why, for the ELISA, did they treat the cells with cycloheximide?
For EGFR degradation using ELISA HEp2 cells were incubated with ligands for 1 h on ice. After the pulse period, cells were rinsed in cold starvation medium and then incubated in warm starvation medium containing 10 μg/mL cycloheximide at 37◦C for0–8 h. Then the cells were rinsed in cold PBS and scraped off in RIPA lysis buffer(1% NP40, 20 mM MOPS, 0.1% SDS, 1% Na-deoxycholate, 150 mMNaCl, and 1 mM ethylenediaminetetraacetic acid) supplemented withProtease Inhibitor Cocktail Set II and Phosphatase Inhibitor Cocktail Set III(Calbiochem). Cell debris was removed by spinning. EGFR content wasthen measured using an EGFR ELISA kit (RnD Systems).
Alternatively, using the pulse-labelassay the cells were pulse- labeled with 35S-methionine, stimulated with ligand for 1 h on ice, washed, and incubated for 2 or 6 h at 37◦ C. EGFR was subsequently immune-precipitated and the amount of 35S-labelled EGFR quantified by Phosphor-Imaging of an SDS-PAGE gel.
The result was that stimulation with TGF-α, EPI, or AR did not lead to significant degradation of EGFR. Stimulation with either EGF or HB-EGF leads to degradation of 40–60% of the cellular EGFR, whereas stimulation with BTC leadsto degradation of approximately 70% of the cellular EGFR.
The two methods differed in that in ELISA cycloheximide was used whereas in the pulse- label assay it was not. In addition, 35S-methionine was used in the pulse-label assay and the cells were incubated for 2 or 6h at 37◦C unlike in the ELISA assay.
Unlike in the pulse-labeled method cyclohexamide was used in ELISA. This was done inorder to inhibit de novo EGFR synthesis.
Q.G. EGFR degradation analysis using ELISA detection
HEp2 cells were incubated with ligands for 1 h on ice. After the pulse period, the cells were rinsed in cold starvation medium and then incubated inwarm starvation medium containing 10 μg/mL cycloheximide at 37◦C for0–8 h. Cells were rinsed in cold PBS and scraped off in RIPA lysis buffer (1% NP40, 20 mM MOPS, 0.1% SDS, 1% Na-deoxycholate, 150 mMNaCl, and 1 mM ethylenediaminetetraacetic acid) supplemented withProtease Inhibitor Cocktail Set II and Phosphatase Inhibitor Cocktail Set III(Calbiochem). Cell debris was removed by spinning. EGFR content wasthen measured using an EGFR ELISA kit (RnD Systems).
Q.H. Describe the general process of how a protein is internalized via endocytosis. Include details about target protein sorting, vesicle formation, and vesicle targeting, and the possible routes for the endocytic vesicle.
Endocytosis is an energy-using process by which cells absorb molecules (such as proteins) by engulfing them. It is used by all cells of the body because most substances important to them are large polar molecules that cannot pass through the hydrophobic plasma or cell membrane. Endocytosis is required for a vast number of functions that are essential for the wellbeing of a cell. It intimately regulates many processes, including nutrient uptake, cell adhesion and migration, receptor signaling pathogen entry, neurotransmission, receptor downregulation, antigen presentation, cell polarity, mitosis, growth and differentiation, and drug delivery. This new book examines the functions and benefits of endocytosis in regards to cells within the body.
Proteins taken into the cell by receptor-mediated endocytosis are transferred to early endosomes where receptors are removed and returned to the cell surface. The proteins are then moved to late endosomes and finally to lysosomeswhere digestion occurs. The lysosomes receive both proteins from the cell surface and digestive enzymes coming from the Golgi.