Physiology and Pharmacology: RTK

Question 1

Structure of RTKs

Answer 1

• Large extracellular domain for ligand recognition and binding
• small alpha helix spanning membrane for information transfer
• Intracellular tyrosine kinase domain -> enzyme activity

Question 2

Pathway of activation for RTK?

Answer 2

Ligand binding causes conformational change

Results in receptor dimerisation (clustering)
(Ligand induced oligomerization)

Intracellular receptor domains can act on each other due to brief conformational change

Transphosphorylation by activation loops on each intracellular domain resulting in activation

Intracellular domains highly active due to newly phosphorylated tyrosine residues -> active kinases

Question 3

How does receptor clustering activate the kinase enzymes?

Answer 3

Signal transduction pathways

• Protein phosphorylation (addition of phosphate)
• Causes structural alteration
• Changes protein activity
• i.e. Active site exposed

Question 4

Explain protein recruitment in the signal transduction pathway

Answer 4

EGF protein -> multiple phosphorylation of kinases
-> phospholipase C 
-> PIP2
-> IP3
IP3 receptors on endoplasmic reticulum
-> Ca2+ flux

Question 5

Describe the direct recruitment and activation of PLC by EGF Receptor tyrosine kinase

Answer 5

• EGF binds to EGFR causing dimerisation, intracellular domain phosphorylation and activation
• Phosphorylated tyrosines are binding sites for recruitment of signalling proteins
• New phosphorylation allows recruitment of Phospholipase C via its SH2 domain to RTK, allowing it to act on PIP2 in membrane
• PIP2 cleaved releasing IP3, leaving DAG in membrane
• IP3 binds to ER calcium channels, opening them and allowing influx of calcium to cytosol

Question 6

What are SH2 domains? How is their specificity determined?

- examples of SH2 domains and target proteins

Answer 6

Modules that direct proteins to specific pY containing targets -> proteins recognise phosphotyrosines

Specificity is determined by residues C-terminal to pY

e.g. PI3 kinase and Grb14

Question 7

How do RTKs recruit multiple binding partners? Function?

Answer 7

RTKs usually have multiple tyrosines
-> can recruit multiple binding partners

Functions

• localise signalling complexes to juxtamembrane regions
• increase efficiency of physical/functional interaction between proteins, permitting regulation of activity/phosphorylation

Question 8

How do proteins which don’t have SH2 domains interact with RTK?
- example of a protein and its interaction

Answer 8

Require adaptor proteins (‘linker’ proteins)
- e.g. SoS with adaptor protein GRB2

GRB2 has both SH2 and SoS binding domains
- Binding of SoS to GRB2 allows SH2 domain binding to pY

Question 9

Which type of g protein family is associated with TRKs?

Answer 9

small G proteins

Question 10

Describe activation of G proteins by RTKs

Answer 10

• Grb2-SoS complex binds to pY via SH2 domain
• now recruited to membrane, SoS acts as a GEF
• > GEF induces GTP -> GDP exchange in RAS protein (small G protein), activating it
• Activated RAS can activate protein kinase RAF
• Kinase cascade
  
  • RAF phosphorylates MEK1 and 2
  • MEK phosphorylate ERK1 and 2
  • ERK phosphorylate transcription factors in nucleus
  • gene expression regulated