Answers
9.(A)
Limitations in the knowledge of the spatial regulation of EGFR signalling imitate the shortcomings of the tools used to block receptor internalization.
Use of either dominant-negative proteins or RNA interference (RNAi) needs that either the dominant-negative construct is expressed or the protein is knocked down for substantial periods of time.
This temporal constraint introduces the option of compensatory mechanisms arising, such as receptor upregulation or changes in the steady-state distribution of the receptor as the cell tries to maintain homeostasis.
On the other hand, some inhibitors of endocytic trafficking, both pharmacologic and biochemical, can be non-specific and interrupt multiple endocytic trafficking actions.
This confounds the explanation of EGFR-specific effects. Finally, several of the approaches do not permit for a satisfactory distinction between variations in the duration of receptor activity and spatial placement of the receptor.
In order to overcome these limitations, Scientists have employed an EGFR-specific ligand that stops internalization of the EGFR, without troublemaking the internalization of other proteins. By using MDA-MB-468 cells that express high levels of EGFRs, have reduced endocytic trafficking, and exhibit no substantial EGFR degradation over time, we can relate the functional significance of EGFR localization and avoid the difficulties of varying levels of the activated receptor.
9(b)
Treatment of EGFR with cycloheximide brings ligand independent internalization of EGFR in 10 minutes, which will not only affect the action of the receptor, but also most likely affect the turnover rate
Phosphorylation can helps in target a protein for degradation and thereby increase its turnover rate. EGFR in wild-type cells was found to have a half-life of around 20 hours, but when EGF was supplemented to the cells, the half-life dropped to about 9 hours.EGFR degradation is observed to primarily happen when the protein is ubiquitinated after ligand-dependent stimulation and phosphorylation. The protein is internalized and sent to endosomes to be manifested for degradation.
Interestingly, internalization alone of EGFR does not consequences in degradation via lysosomes and could possibly delay degradation of the protein by possession it from being activated
Amino acid reprocessing is an vital aspect to study when calculating protein turnover rates. Study anticipated that the favored amino acid supply for protein consolidation is from the extracellular pool and not amino acid recycling. Due to no cleavages during the enzymatic digestion step, the integration of both labeled (new) amino acids and unlabeled (recycled) amino acids in the same peptide can be estimated.
The occurrence of both types of amino acids in one peptide only arises when unlabeled amino acids were used, providing a method to observe amino acid recycling. Turnover rates calculated by only accounting for peptides with completely heavy and completely light lysine (K) and arginine (R) miscalculate the actual turnover rate.
Protein degradation duties can be used to estimate protein turnover, although using degradation rates alone most likely results in an extra estimation of the turnover time. EGFR degradation in A431 cells was previously measured by Stoscheck and Carpenter, who found the rate of EGFR degradation in A431 cells to have = 20 hrs.
This rate was calculated by labeling the receptor in vitro with radioactive amino acid precursors and chased with nonradioactive media. Other measurements of EGFR half-lives in the absence of EGF range from 10 to 48 hours
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