RTKs

Question 1

1. what must an amino acid contain in order to be phosphorylated?
2. which 3 amino acids meet this critera?

Answer 1

1. a free hydroxyl (-OH) group
2. tyrosine, threonine and serine

Question 2

1. how many proteins make up an RTK? Which is an exception to this and why?
2. what domains (2) do ligand binding sites contain?
3. ligands of RTKs are typically what?
4. Name the 8 RTKs and some of the effects that they mediate

Answer 2

1. a single polypeptide. Insulin is an exception because it consists of 2 polypeptides that are linked by a disulphide bond
2. Ig and Fibronectin domains
3. peptide growth factors
4. Eph receptor - mediates axonal migrationEGFR - mediates proliferationInsulin receptor - mediates protein synthesis and carbohydrate utilisationinsulin-like growth factor receptor - mediares cell growth and survival; implicated in longevityPDGFRNGFR - required for neuronal survival during developmentFGFRVEGFR - vasculogenesis

Question 3

1. Describe the process of receptor activation
2. state 3 ways in which dimerisation is acheived.

Answer 3

1. ligand binding causes dimerisation. Dimerisation causes 2 tyrosinke kinase domains to come into close proximity with one another leading to cross phosphorylation.

cross phosphorylation causes kinase activity to increase, and creates p-Tyr docking sites for SH2/PTB containing proteins.

2. a monomeric ligand induces a conformational change in the receptor which induces dimerisation

the interraction of the ligand with a disulphide established receptor dimer

mediation of dimerisation through a dimeric ligand

Question 4

1. What is each peptide of the insulin receptor made up of?
2. What is IRS1?

Answer 4

1. alpha and beta subunits
2. a protein which contains a PTB domain allowing it to be recruited to the activa receptor. It is essentially a scaffold, as when docked at the receptor, it is phosphorylated, creating more p-tyr docking sites

Question 5

describe how the insulin receptor structure was determined.

Answer 5

receptors were engineered to contain a gold particle at a fixed position. Following visualisation by Cryo-EM microscopy at different postions, the outline of the receptor was established (by looking at where the gold particle lay)

each domain was then separately crystalised. Using the outline of the receptor established by Cryo-EM, how these domains were organised was determined.

Question 6

1. what is the structure of the tyrosine kinase domain of the insulin receptor?
2. how is the activity of this domain regulated?

Answer 6

1. c-lobe, regulatory loop and n-lobe. The regulatory loop contains an inhibitory tyrosine, and when the receptor is active, forms the substrate binding domain
2. when inactive, the regulatory loop is buried inside the c-lobe, preventing substrate bindingcross-phosphorylation involves the phosphorylation of the inhibitory tyrosine, which causes a conformational change in the regulatory loop, enabling it to pop out of the C-lobe and bind to substrates.

Question 7

How does an SH2 domain interract with p-Tyr?

Answer 7

It contains 2 arginines and a lysine. One arginine and the lysine interract with the aromatic rings of the tyrosine. The second arginine interracts with the negatively charged phosphate to distinguish between p-tyr and normal tyr. (co-incidence detection is used to distinguish p-Tyr from normal Tyr)

Question 8

1. describe how Activation of RTK is transduced to Ras
2. describe a novel model of Ras activation involvong phospholipase D2
3. How does Ras activate MAPK?
4. How does MAPK mediate survival and proliferation?
5. How is signal transduction amplified?

Answer 8

1. SH2 domain of GRB2 interracts with p-Tyr on receptorSH3 domain of GRB2 recruits Sos (a Ras GEF)Sos activates Ras which is located at the membrane
2. PLD2 is located at the membrane and catalyses the hydrolysis of phosphatidyl choline into phosphatidic acid

Sos has a PH domain therefore undergoes high affinity binding to PA

PLD2 also binds to GRB2.

3. Ras > MAPKKK > MAPKK > MAPK
4. MAPK translocates to the nucleus where it regulates the stability of transcription factors such as c-fos and c-jun by phosphorylation
5. it is an enzyme cascade - one enzyme can act on many substrates therefore amplification is exponential

Question 9

How does the PI₃ kinase mediated production of PI(3)P lead to cell survival?

Answer 9

PI(3)P serves as a docking site for the PH containing Akt.

Akt is therefore recruited to the membrane, where it is phosphorylated by mOTR and PKDI, thus is activated

Active Akt dissociates from the memrane and phosphorylates Bad

p-Bad releases proteins that inhibit apoptosis.

Question 10

1. why is co-incidence binding sometimes needed for the recruitment of adaptor protiens to the membrane?
2. give an example of a protein that undergoes co-incidence binding

Answer 10

1. because the interraction between SH2 and p-tyr is of low affinity
2. PLC-gamma which undergoes co-incidence binding with p-Tyr and PI(4,5,6)P

Question 11

1. in general, how is the specificity of signalling acheived?
2. Using NGF and EGF as an example, describe how specificicty of signals that act through the same pathway is achieved.

Answer 11

1. positive and negative feedback loops
2. EGF application to PC12 cells > proliferation; NGF application to PC12 cells > differentiationthe specificity of the signalling outcome is mediated by the kinetics of MAPK activation. (EGF transiently activates MAPK, while NGF mediated MAPK activation is sustained)

this is mediated by trafficking, feedback loops and the regulation of transciption

TRAFFICKING
- EGFR is endocytosed and trafficked to the lysosome for degradation

• NGFR is endocytosed and recycled

FEEDBACK LOOPS
-EGF mediated MAPK actvation is regulated by negative feedback loops as MAPK inhbits MAP-KKK

• NGF mediated MAPK activation is regulated by positive feedback loops as MAPK stimulates MAP-KKK

TRANSCRIPTION REGULATION
- because EGF only transiently activates MAPK, it is unable to enter the nucleus and phosphorylate TFs such as c-Fos. As a result, c-Fos isnt stabilised, thus is degraded

• EGFR activation also leads to the transcription of phosphatases that dephosphorylate S/T kinases (MAPKK)
• NGFR activates MAPK sufficiently for it to be translocated to the nucleus where it phosphorylates thus stabilises c-Fos so that it can mediate transcription.

Question 12

1. name 4 ways that mediates abberant RTK activation in cancer.
2. name 2 types of drug that have been developed to disrupt overactive RTKs
3. How is HER2 a positive regulator of EGF signalling?

Answer 12

1. RTK overexpression; RTK GOF mutations; autocrine activation; chromosomal translocations (loss of regulatory loop?)
2. small molecule inhibitors; monoclonal antibodies
3. It doesnt bind a ligand, and is always in an active conformation to dimerise with another receptor. It therefore amplifies and prolongs signalling by forming heterodimers with ligand occupied receptors.

Question 13

1. what are antibody drug conjugates.
2. give 2 ways in which antibodies can treat cancer?

Answer 13

1. an antibody that is conjugated with a drug, that is used for the selective delivery of cytotoxic agents to tumours.
2. one antibody sequesters VEGFA therefore slowing down the formation of tumour vasculatureanother antibody used to treat non-hogkin’s lymphoma, drives the oncogenic B cells they bind to towards apoptosis

Question 14

1. describe the 3 methods used to show that FGFR1 and 2 are involved in would healing
2. what were the findings of these experiments?

Answer 14

1. generation of mice lacking either FGFR1, FGFR2 or bothgenerated full thickness wounds and observed the healing processlooked at the migratory and adhesion capacity of keratinocytes
2. only mice lacking both receptors had impaired wound healingFGFR1/2 deficient keratinocytes had reduced migration rate with impaired directionalityFGFR1/2 deficient keratinocyte adhesion was reduced (they had fewer focal adhesions than controls; reduced expression of FA kinase and paxillin)

Question 15

1. How was the mechanism of VEGF2R induced arterial morphogenesis elucidated?
2. what were the results of these experiments?

Answer 15

1. KD of synectin and myosin V in vitro and in vivoVEGFR activation was examined by western blottingexamined proteins that bound to synectin
2. decreased MAPK activation, VEGF2R Y1175 phosphorylation and PLCgamma phosphorylation in KD cells

delayed internalisation of VEGF2R in synectin and myosin V KD

in vivo myosin KD caused similar defects seen in vitro plus impaired vasculogenesis

KD of p-tyrosine phosphatase restored ERK activation and Y1175 phosphorylation in KD

VEGF binding > VEGFR phosphorylation > activation > autophosphorylation > endocytosis > binding of synectin-myosin complex > movement to EEA1 positive endosomes > Activation of MAPK/PLC-gamma/Akt > arteriogenic signalling