Growth factor receptors and their signalling pathways Flashcards
What are Growth factors ?
- Growth factors are a variety of factors which have specific functions in the regulation of growth and differentiation of cells
- Nerve growth factor (NGF) first discovered. Major ones include EGF, FGF, VEGF, IGF
What is the signalling for Growth factors ?
Generally paracrine (act locally, not produced by specialised tissues) but can be autocrine. Peptide molecules
Explain Epidermal Growth Factor ?
- Cellular changes include induction of ornithine decarboxylase and increased polyamine concentrations (↑ rate of cell division)
- In culture EGF increases the rate of cell growth and division: Increased synthesis of DNA, RNA and protein
- General anabolic effects similar to insulin e.g. increased transport of glucose and amino acids, purine and pyrimidine precursors, ↑ Na+/K+ ATPase
- Increased Na+ entry (Na+/H+ exchange)
- Increased alkalinisation of cytoplasm (↑ DNA synthesis/mitosis)
- Increased cytosolic Ca 2+ and PKC activity
Who do all growth factors signal via ?
- Receptor tyrosine kinases: two receptor molecules dimerise on ligand binding and cross-phosphorylate each other
- All have a tyrosine kinase domain on cytoplasmic side of the plasma membrane (blue)
- Extracellular domain is unique
Explain binding of EGF1 to its receptor ?
- Dimerisation generates an asymmetric kinase dimer in which the donor kinase C-terminal C-lobe binds the acceptor kinase N-terminal N-lobe
- This causes a conformational change that removes the activation loop from the acceptor kinase site, activating its kinase activity
- The active acceptor kinase then phosphorylates cytosolic domain tyrosine residues on both receptors
Explain the activation of Receptor Tyrosine Kinases ?
- Binding of ligand induces receptor dimerisation
- Receptors cross-phosphorylate on activation loop of tyrosine kinase domain
- This then fully activates enzyme activity, and it phosphorylates on additional tyrosine residues: this creates docking sites for proteins with SH2 domains to bind
Explain the activation of Ras by Receptor ?
- Binding of ligand causes receptor dimerisation and autophosphorylation
- The activated receptor recruits adaptor proteins
- SH2 domain in GRB2 binds to phosphorylated tyrosine residues in receptor
- SH3 domain of GRB2 binds to proline-rich regions of Sos (a GEF) and recruits to membrane
- This couples receptor to inactive Ras
- Sos, the guanine nucleotide exchange factor (GEF) promotes dissociation of GDP from Ras
- GTP binds and GEF dissociates from active Ras
- This then activates downstream signalling
How can Ras be activated ?
Activation of Ras by Guanine Nucleotides
What is Ras ?
Ras is a small G protein (similar to an alpha subunit of a heterotrimeric G protein)
What does GEF stand for ? and what does it promote ?
GEF – Guanine nucleotide Exchange Factor:
- Promotes exchange of GDP for GTP, i.e. swapping molecules
What does GAP stand for ? and why do you need GAP ?
- GAP = GTPase activating protein
- Need GAP to keep turning over at higher rate
Third phosphate in GTP acts as ? (Ras as a molecular switch)
- A connection between switch 2 and switch 3 regions
When GDP bound, the switch regions ?
Spring open with no phosphate to connect them
How is Ras also activated ? and explain ?
- GEF
- An alpha helix from the GEF goes into the binding pocket of Ras and displaces the GDP
- GTP, which is at much higher concentrations in the cell, is now able to bind
What family is ERK part of ?
ERK is in the MAPK family (mitogen-activated protein kinases)
- specific for Ser or Thr residues
What family is MER part of ?
MEK is in the MAP kinase kinase (MAPKK) family
- activates the MAP kinase
- phosphorylates both Ser and Tyr residues
What family is Raf-1 part of ?
Raf-1 is in the MAP kinase kinase kinase (MAPKKK) family
- activates the MAP kinase kinase
- specific for Ser or Thr residues
Ras-GTP recruits ?
The protein kinase, Raf (MAPKKK), to the membrane
Interaction with Ras-GTP relieves ?
Inhibition by 14-3-3 protein, leading to dephosphosphorylation of the regulatory domain
C-terminal catalytic domain of Raf is then phosphorylated by?
Src kinase and activated
Activation of Raf (serine/threonine kinase) results in ?
The binding and phosphorylation of the protein kinase MEK (MAPKK), activating it
MEK recruits and phosphorylates ?
MAP kinase (ERK), on both tyrosine and threonine residues leading to its activation
What is MAPK ?
MAPK is a serine/threonine kinase that phosphorylates downstream target proteins
You are studying a cancer cell line that causes uncontrolled proliferation of cultured fibroblasts. You have identified some candidate mutations in an RTK pathway. Which mutation(s) could cause this effect?
- A mutation that inactivates the phosphatase which normally removes phosphates from the receptor, will mean it stays switched on, inappropriately stimulating cell proliferation
- A mutation that prevents Ras from hydrolysing GTP will keep it constitutively active: therefore downstream activity will stimulate cell proliferation regardless of presence of ligand
What does MAP kinase regulates ?
MAP kinase regulates the activity of many transcription factors controlling the ~1100 early response genes activated by adding a growth factor to quiescent cells
