Tyrosine Kinase receptor signalling 1

Question 1

Name 3 Enzyme-linked cell surface receptors and examples

Answer 1

1. Receptor tyrosine kinases (RTKs) – class/super family
   e. g. FGFRs, PDGFRs - family
2. Tyrosine-kinase-associated receptors – class/super family
   e. g. cytokine receptors- family
3. Receptor serine/threonine kinases – class/super family
   e. g. TGFBRs, BMPRs - family

Question 2

What is a cell culture

Answer 2

1. Much of what we know comes from experiments in cell culture.
2. Different mammalian cells need the same basic conditions
3. (buffered medium, nutrients , O2, temp) and also SERUM, which can be replaced with specific signal molecules, needed for survival, proliferation and differentiation.
4. Most cells are grown as adherent cultures in plastic flasks, dishes or multiwell plates. – on a flat surface

Question 3

What are different growth factor signal proteins

Answer 3

1. Epidermal growth factor (egf)
2. Insulin-like-growth factor 1 (lgf1)
3. Fibroblast growth factors
4. Platelet derived growth factor

Question 4

What do many “growth factors“ ligands signal via

Answer 4

1. Many “growth factors“ ligands signal via RTKs

Question 5

How can signalling influence growth

Answer 5

Signalling can influence growth at the level of tissues, organs and the whole body through regulation of:

1. Cell survival,
2. proliferation,
3. differentiation and growth (size)

Question 6

How is growth defined

Answer 6

1. Strictly, at the cellular level growth is an increase in cell size (volume), whereas tissue, organ or whole body growth can involve increases in cell size, number and also extra-cellular matrix/fluid.
2. Growth during development or tissue renewal also involves a balance between cell proliferation and differentiation.

Question 7

How does Insulin-like growth factor signalling regulate fetal growth

Answer 7

1. Two ligands interact with one receptor
2. IGF1 and IGF2 interact with IGF1R to stimulate growth during development
3. We know about the functions of the IGF signalling system in promoting proliferation and survival of many cell types,
4. first from cell culture experiments
5. then from mouse gene knockout studies.
6. In addition, cell number is a balance between production and loss (turnover).
7. IGFs are also important for human growth and development

Question 8

What are associated with mutations of IGFs

Answer 8

1. Rare growth restriction syndromes are associated with mutations
2. inactivating the IGF1R gene (leprechaunism)
3. with loss of IGF2 gene expression (Silver Russel Syndrome)
4. while IGF2 excess occurs in the overgrowth disorder, Beckwith Weidemann syndrome and in cancers.

Question 9

What are characteristics of RTKs

Answer 9

1. Cysteine-rich extracellular domain
2. Single a-helical hydrophobic transmembrane domain - Allow it to spam plasma membrane
3. Intracellular domain with tyrosine kinase activity and several tyrosine residues
4. Phosphorylation is a key mechanism in regulating protein function; phosphate group is an effective allosteric activator.
5. Rapid phosphorylation of RTKs themselves is a characteristic of RTK signalling.

Question 10

What are families

Answer 10

1. growth factors and their receptors are found in structurally related families.
2. The closely related insulin receptor and IGF1R offer a clue to understanding how these receptors function.
3. 4 subunits
4. 2 identical which spam plasma membrane- containing kinase
5. 2 identical which comprise extracellular domain

Question 11

Describe an experiment to investigate function of the EGFR kinase domain

Answer 11

1. EGFR (Epidermal growth factor receptor) (or FGFR) are introduced into cells using a mammalian expression vector, such that the gene is transcribed under the control of an appropriate promoter,
2. the receptor protein translated and localised correctly in the plasma membrane.
3. The mutant EGFR has a point mutation that inactivates the kinase.
4. FGFR- used as control so no response as doesn’t recognise ligand
5. Point mutation- see importance of different parts

Question 12

Describe Receptor mediated endocytosis

Answer 12

1. Over time receptors that are bound to ligand are incorporated into clathrin coated pits which are pinched off into vesicles which then fuse with endosomes
2. Endosomes have low pH 5-6
3. Ligand tends to dissociate from receptor
4. These endosomes fuse back to cell surface
5. Receptors are recycled
6. Endosomes can fuses with lysosomal compartments
7. Conditions pH is even lower
8. Conditions mean lysosomal enzymes can digest proteins- receptor and ligand
9. So level of receptor is reduced over time even though there is some recycling
10. Makes it harder for ligand
11. Process doesn’t propagate signal
12. Allows the signalling system to adapt to chronic exposure to signal so harder for signal to get propagated

Question 13

How could you modify the experiment to identify regions of the EGFR extracellular domain required for ligand binding?

Answer 13

1. In this case you could mutate or delete one or more amino acids in the extracellular domain to see which abolish the response.
2. Similar mutation analyses have been valuable in combination with human genetic studies, in which mutations found in people with growth disorders (often cancers) are copied to study the functional outcome.
3. Activating mutation in RTKs are a common feature of many cancers.

Question 14

How does the signal cross the membrane in transduction through the membrane

Answer 14

1. RTKs achieve signal transduction through dimerisation/oligomerisation and subsequent autophosphorylation.
2. Signal transduction through a single transmembrane domain is unlikely (compare with G-protein coupled rec.) because the plasma membrane phospholipid bilayer acts as an insulating barrier between the extra- and intra-cellular domains.
3. The signal (ligand) interacts with the receptor with high affinity and specificity to bring together two copies of the receptor.

Question 15

What dimers can the alpha and beta receptor form

Answer 15

1. the alpha receptor could form dimers with any of the three PDGF (platelet derived growth factor) dimers, AA, AB or BB
2. whereas the beta receptor can only form dimers with PDGF-BB.
3. Ligands interact with receptors alpha and beta
4. Alpha - A and B
5. Beta – only interact with B

Question 16

What is the first key experiment proving the importance of receptor dimerization for RTK activation.

Answer 16

A form of ligand
1. doesn’t interact with beta
2. Respond strictly with A form
3. Tyrosine kinase activity
4. Dna synthesis 
5. PDGFRa internalisation
AB - depletion of alpha receptor
1. Only b part interacts with beta receptor
2. No tyrosine kinase activity
3. No DNA synthesis
4. Shows need for dimer to interact
BB
1. Both parts interact
2. tyrosine kinase activity
3.Dna synthesis
4. Receptor internalisation

Question 17

What is the second key experiment proving the importance of receptor dimerization for RTK activation.

Answer 17

1. Kinase inactivating, dominant-negative, mutations are found in, e.g. The KIT receptor (piebaldism); INSR (severe, inherited diabetes and growth restriction).
2. Activating mutations (kinase activity in the absence of signal) are seen in, e.g. in cancers (various RTKs), and in achondroplasia (FGFR3*)

Question 18

What are the Substrates of tyrosine kinase domain

Answer 18

1. Phospholipase C-gamma
2. Src
3. PI3-kinase
4. GAP
5. These proteins contain conserved binding motifs
6. SH2 domain (Src homology region) for receptor interaction
7. SH3 domain for interactions with other signalling proteins

Question 19

Describe activation of RTKs such as PDGF-R

Answer 19

1. Activation of RTKs such as PDGF-R involves autophosphorylation
2. Dimerisation mediated by ligand binding brings two receptor molecules together, allowing one kinase domain to phosphorylate the tyrosine residues within the kinase domain of the other receptor.
3. This phosphorylation event activates the kinase, raising the level of activity such that each kinase can add phosphates to further tyrosine residues on the opposing receptor’s intracellular domain.

Question 20

Describe RTK activation of SH2/SH3 domain proteins

Answer 20

1. Phosphorylation of the receptor results in conformational changes to the domains surrounding each tyrosine residue.
2. These shape changes allow specific interactions with multiple intracellular proteins via SH2 domains.
3. SH3 domains allow further interactions with non-receptor signalling proteins.

Question 21

Why are RTKs important targets for new drug molecules

Answer 21

1. Deregulation of RTK signalling is crucial for the development of hyperproliferative diseases, e.g. cancer.
2. Advanced small molecule inhibitors (e.g. Osimertinib) target the mutant forms of kinases commonly found in cancers while leaving the wild type receptor unaffected.