Lecture 3 Growth Factor Receptors Flashcards
Tyrosine phosphorylation was seen to massively increase in cells incubated with EGF and radioactive phosphate. What is significant about this tyrosine phosphorylation
The majority of the increase in tyrosine phosphorylation in response to growth factors was occurring within the receptor itself. This is due to the oligomerisation of receptor monomers and subsequent auto and cross-phosphorylation
Proto-oncogenes that are growth factor receptors all possess and intracellular domain with tyrosine kinase activity T or F
F – the JAK/STAT receptors for example for α-interferon have a separate protein that possesses the kinase activity
What does this data tell us about the significance of PTB domain containing protein localisation in response to growth factors
Immunofluorescent staining was used to show the localisation of PTB domain containing proteins along with the EGF receptor. EGFR expression was localised to the membrane shown with a high intensity sharp outline of the cell representing the EGFR protein in the membrane. However in the absence of EGF ligand PTB domain contain proteins were present in the cytosol. Upon addition of EGF into the medium this staining changed and a sharp bright outline was seen. This corresponded to the localisation of PTB proteins to the membrane which actually co-localised with the EGFR.
It was found that proliferation of cells in vitro requires serum how can this be mimicked. What is the significance of this
Purified growth factors fulfil the serum requirements of cells. This indicated that growth factors may be involved in triggering proliferation through a mechanism that may involve protein phosphorylation
How was it determined that src was a kinase
Collett and Erikson developed an antibody against the src protein. This antibody was then attached to beads in a column before an immunoprecipitation was carried out to elute the src protein. This was done by passing the lysates from control cells and those who had been infected with Rous sarcoma virus through the column one population from each had been injected with the anti-src antibody. The src protein eluted from each lane was then incubated with radioactive ATP (γ32P-ATP) to test whether it would be incorporated into a substrate seeing as it was already known that ATP was a substrate for protein kinases. The transformed cells incubated with radioactive ATP possessed an additional band indicating the phosphorylation of a substrate protein with the radioactive ATP. This determined that src was indeed a protein kinase
Notch/delta receptors are also proto-oncogenes. How do these differ from other proto-oncogenes
Notch/delta receptors are non-identical receptors unlike the RTKs. In addition they don’t possess an intracellular kinase domain. Instead binding of Notch to delta causes proteolysis of the cytoplasmic domain which then translocated to the nucleus to direct transcription. Finally the signalling from these proto-oncogenes is irreversible in comparison to RTK signalling where tyrosine phosphorylation can be reversed by phosphatases
How was the transmembrane domain of EGFR identified
A hydropathy plot was carried out on the amino acid sequence of the EGFR protein. Here rolling groups of 10 amino acids were used to determine the hydrophobicity/lipophobicity. One large peak was observed corresponding to a highly hydrophobic region of the protein. This was later found to be the transmembrane domain
What was the conceptual framework for the mechanism of transformation that was proposed as a result of work carried out on src and polyoma large T
That kinases such as src act as signalling proteins that phosphorylate tyrosine residues in target proteins to cause a change in function
Explain the experiments that determined which amino acid was being phosphorylated by src
It was found that the polyoma large T protein was a transforming viral protein that was also being phosphorylated by src. This protein was digested into its constituent amino acids which were then separated in two planes using electrophoresis (to separate based on charge) in the x-axis and chromatography (to separate based on size) in the y-axis. It was expected that there would be specific phosphorylated residues representative of serine or threonine however instead a region between these two regions was observed. This was indicative of tyrosine and hence lead to the idea that src was phosphorylating tyrosine residues in the polyoma large T protein
How was the tyrosine kinase domain of EGFR identified
The amino acid sequence of EGFR was compared against the known sequences of the time (<100). Surprisingly the C-terminal fragment was found to have a high degree of homology to src. This was already known to be a tyrosine kinase conferring the idea that the EGFR receptor was indeed also a tyrosine kinase
What were the subsequent conclusions from work carried out by Cohen characterising EGFR. How does this relate to oncogenes
Growth factors induce their cellular effects by triggering a tyrosine kinase signalling pathway. Oncogenes might work by triggering signalling pathways in the absence of appropriate extracellular cues
How was the ligand binding domain of EGFR characterised
Functional analysis of the EGF protein was carried out. The protein was cut at specific positions using chemical to digest it this generated a small number of large fragments. These fragments were then run on a gel before being transferred to a membrane and the blot probed with radioactive-EGF. Bands where radioactivity was observed indicated regions of the protein that bound EGF. It was found that the large N-terminal fragment band bound radioactive-EGF. This was later found to be the ligand binding domain
What was significant about what was being phosphorylated by src in the immoprecipitation experiments carried out by Collet and Erikson
It turned out the what was being phosphorylated was the anti-src antibody. This was completely by chance
Give an example of how the signalling from receptors that are proto-oncogenes can differ
Some signalling is reversible such as phosphorylation of amino acids in the case of receptor tyrosine kinases whereas others are non-reversible such as the cytoplasmic cleavage of the notch receptor.
Describe the experiments of Frackelton that determined that growth factor receptors are tyrosine kinases
Frackleton incubated cells in the presence of radioactive phosphate containing the 32P isotope either with EGF present or absent from the medium. Time was allowed for the cells to take up the radioactive phosphate which incorporated into the cells proteins. The protein lysates from these cells were then digested into their constituent amino acids before they were separated in two dimensions using electrophoresis and chromatography. Once the amino acids had been separated they were exposed to film which changed colour depending on radioactivity. Phosphothreonine and phosphoserine residues were identified at comparable levels in both the cells incubated with and without EGF. However an additional band increased massively in the cells incubated with EGF. This band corresponds to a massive increase in tyrosine phosphorylation as a result of EGFR activation.
