Growth factor receptors and their signalling pathways

Question 1

What are Growth factors ?

Answer 1

• 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

Question 2

What is the signalling for Growth factors ?

Answer 2

Generally paracrine (act locally, not produced by specialised tissues) but can be autocrine. Peptide molecules

Question 3

Explain Epidermal Growth Factor ?

Answer 3

• 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

Question 4

Who do all growth factors signal via ?

Answer 4

• 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

Question 5

Explain binding of EGF1 to its receptor ?

Answer 5

• 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

Question 6

Explain the activation of Receptor Tyrosine Kinases ?

Answer 6

• 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

Question 7

Explain the activation of Ras by Receptor ?

Answer 7

• 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

Question 8

How can Ras be activated ?

Answer 8

Activation of Ras by Guanine Nucleotides

Question 9

What is Ras ?

Answer 9

Ras is a small G protein (similar to an alpha subunit of a heterotrimeric G protein)

Question 10

What does GEF stand for ? and what does it promote ?

Answer 10

GEF – Guanine nucleotide Exchange Factor:

- Promotes exchange of GDP for GTP, i.e. swapping molecules

Question 11

What does GAP stand for ? and why do you need GAP ?

Answer 11

• GAP = GTPase activating protein

- Need GAP to keep turning over at higher rate

Question 12

Third phosphate in GTP acts as ? (Ras as a molecular switch)

Answer 12

• A connection between switch 2 and switch 3 regions

Question 13

When GDP bound, the switch regions ?

Answer 13

Spring open with no phosphate to connect them

Question 14

How is Ras also activated ? and explain ?

Answer 14

• 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

Question 15

What family is ERK part of ?

Answer 15

ERK is in the MAPK family (mitogen-activated protein kinases)
- specific for Ser or Thr residues

Question 16

What family is MER part of ?

Answer 16

MEK is in the MAP kinase kinase (MAPKK) family

• activates the MAP kinase
• phosphorylates both Ser and Tyr residues

Question 17

What family is Raf-1 part of ?

Answer 17

Raf-1 is in the MAP kinase kinase kinase (MAPKKK) family

• activates the MAP kinase kinase
• specific for Ser or Thr residues

Question 18

Ras-GTP recruits ?

Answer 18

The protein kinase, Raf (MAPKKK), to the membrane

Question 19

Interaction with Ras-GTP relieves ?

Answer 19

Inhibition by 14-3-3 protein, leading to dephosphosphorylation of the regulatory domain

Question 20

C-terminal catalytic domain of Raf is then phosphorylated by?

Answer 20

Src kinase and activated

Question 21

Activation of Raf (serine/threonine kinase) results in ?

Answer 21

The binding and phosphorylation of the protein kinase MEK (MAPKK), activating it

Question 22

MEK recruits and phosphorylates ?

Answer 22

MAP kinase (ERK), on both tyrosine and threonine residues leading to its activation

Question 23

What is MAPK ?

Answer 23

MAPK is a serine/threonine kinase that phosphorylates downstream target proteins

Question 24

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?

Answer 24

• 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

Question 25

What does MAP kinase regulates ?

Answer 25

MAP kinase regulates the activity of many transcription factors controlling the ~1100 early response genes activated by adding a growth factor to quiescent cells