S2: Signalling in Metabolic Regulation

Question 1

Describe growth factors receptors

Answer 1

• Mostly single transmembrane domain receptors
• Activation leads to activation of a receptor kinases
• Activation leads to activation of multiple signalling pathways
• Often activated by a series of phosphorylations or dephosphorylations e.g. tyrosine kinase

Question 2

What are the different types of growth factor receptors?

Answer 2

1. Tyrosine kinase receptor

• Insulin
• Epidermal growth factor (EGF)
• Platelet derived growth factor (PDGF)

2. (JAK/STAT pathway) e.g. Interliekin
3. Receptor Serine/Threonine Kinase - transforming growth factor (TGFB)

Question 3

What does tyrosine kinase help regulate?

Answer 3

• Cell growth
• Division
• Differentiation
• Survival
• Migration

Question 4

Describe activation of Receptor Protein Tyrosine Kinases

Answer 4

Binding of molecule with receptor leads to dimerization (conformational change) that allows it to bind to another receptor molecule so the two receptors are brought into close contact and they can interact leading to activation and autophosphorylation (carried out by neighbouring molecule). This is a transient interaction.

Activation of tyrosine kinase receptors activates multiple pathways

Question 5

Structure of Receptor Protein Tyrosine Kinases

Answer 5

• Single transmembrane domain proteins (exception of insulin)
• Binding site on extracellular domain
• Enzyme activity on cytosolic part of the molecule
• Binding of an agonist to the receptor result in changes in the intracellular domain enabling it to activate a range of different pathways.

Question 6

What are the two ways dimerisation of tyrosine kinase receptors can be done?

Answer 6

Ligand mediated – Could be that agonist binds to one receptor and another to the other and the two agonists come together

Receptor mediated – Could be that binding of agonist causes conformational change in molecules leading to dimerization

These are not covalent interactions, they are transient, the process allows the bringing together of the tyrosine kinase activities associated with the intracellular part of the receptor.

Question 7

How does the tyrosine kinase receptor autophosphorylate the adjacent tyrosines?

Answer 7

Two EGF (epidermal growth factor) bind each tyrosine kinase molecules and then they come together.

The tyrosine activity will then only phosphorylate tyrosines (AA in chain) that are in a specific context/sequence (motif). There may be multiple of these sequences in the intracellular domain, so there may be multiple phosphorylations (so one receptor can be phosphorylated many times).

Question 8

What does the phosphorylated tyrosine kinase receptor then form?

Answer 8

• It will form part an SH2 domain motif.
• This is a site that is recognised by other proteins (with an SH2 domain), allowing other proteins to be recruited to the receptor.
• This recruitment can lead to their direct activation or that it facilitates the recruitment of further proteins.

Proteins containing the SH2 domain will bind to the SH2 domain motif which will then set of a cascade of events.

Question 9

Describe the cascade of events when SH2 domain binds Grb-2 (activation of proteins)

Answer 9

The Grb-2 proteins bind to the SH2 domain motif, then the Grb-2 also through SH2 domains activates another protein Sos, which itself then activates another protein.

This allows very fine regulation because you can regulate any one of these proteins to modulate the end result.

The RAS protein (associated with cancer) which a type of G-protein, binds GDP when inactive. On activation it exchanges the GDP for GTP and then is activated.

Question 10

How can tyrosine kinase activity be regulated?

Answer 10

Mechanism of RAS

• Inactive GDP bound to Ras
• Exchange of GDP to GTP when protein is activated
• This recruits other proteins such as RAF
• The whole process is aided (sped up) by the protein guanine nucleotide exchange factor (GEF)

The GTP binding proteins have GTPase activity, which remove phosphates of the RAS protein, inactivating it. However this is usually very slow. But, there is a protein that aids this called GAP (GTP-activating protein), which helps in the removal of the phosphate.
The cell has this in order to down-regulate this process so there isn’t overstimulation of the cell.

So GAP enhances inactivation. Once it has removed the phosphate, the complex falls apart and then waits in the membrane for further activation.
So this is one of the means by which receptor tyrosine kinase activity can be regulated.

Question 11

Why is the RAS protein important?

Answer 11

It is implicated in some cancers

Mutated Ras is found in 30% of tumours

Question 12

What binds to receptor tyrosine kinase to activate it?

Answer 12

EGF - Epidermal Growth Factor

Question 13

How are receptor tyrosine kinase (RTK) inactivated?

Answer 13

Dephosphorylation leads to inactivation and phosphatases do this.

These phosphatases are activated as a result of the receptor activation and in some cancers the genes for these enzymes are mutated (-> prolonged activation).

Question 14

How is Ras inactivated?

Answer 14

Small g-proteins (such as Ras) have intrinsic GTPase activity so auto-regulate themselves.

Question 15

EGF Receptor Family and Cancer

Answer 15

• Failure to regulate EGF receptor activity is implicated in cancer
• If EGF receptor is mutated and can dimerise in absence of EGF, then it will no longer be dependent on EGF.
• Ras may lose its GTPase activity, so it can no longer turn off its activity, leading to inappropriate growth
• Loss of phosphatase activity means you cannot inactivate the receptor, leading to excessive stimulation of the downstream pathways

Question 16

What is Herceptin?

Answer 16

Monoclonal antibody that targets the EGF receptor blocking its action

Question 17

Describe the structure insulin receptor

Answer 17

The insulin receptor is also a tyrosine kinase type receptor but it has a different format. It consist of two tyrosine kinase receptor molecules that are linked, so it is already a dimer.
- Consists of two beta chains and alpha chain linked by disulphide bridges

Question 18

What is a ECF receptor?

Answer 18

A specific tyrosine kinase receptor

Question 19

Describe mechanism of insulin receptor

Answer 19

• When insulin binds, there is a conformational change which brings together the two beta chains. The beta chains have tyrosine kinase activity so can auto-phosphorylate tyrosine residues on the adjacent beta chain.
• They recruit insulin receptor substrate (IRS). This is a protein recruited through the phosphorylated tyrosines on the beta chain with SH2 domains on the IRS, which then leads to phosphorylation. Causing generation of more phosphorylated tyrosines which can then go on to recruit more proteins.

Question 20

What does insulin receptor substrate (IRS) do?

Answer 20

IRS activates PI3K, which leads on to activate protein kinase B. PKB can then do a number of things including stimulating glycogen synthesis, protein synthesis and increased expression of GLUT transporters on cell surface membrane.

The insulin receptor also recruits other proteins such as Ras and PLC (involved in generation of IP3)

So the insulin receptor is on liver, adipocytes and muscle and helps reduce blood glucose.

Question 21

What do Receptor Tyrosine Kinase primarily control?

Answer 21

Cell growth and differentiation

Question 22

How are growth hormone receptors different to tyrosine kinase receptors?

Answer 22

Differently to the tyrosine kinase receptors spoken about, growth hormone receptors do not have intrinsic tyrosine kinase activity within the intracellular domain. Note they are important for cytokine binding.

Question 23

What activates the Jak-STAT pathway (GH receptor)?

Answer 23

Growth Hormone

Question 24

Mechanism of growth hormone receptor

Answer 24

Binding of growth hormone to its receptor leads to dimerisation, but here only one growth hormone molecule needs to bind to facilitate dimersiation/activation of the receptor.

So this activated growth hormone receptor does not have the ability to phosphorylate tyrosines on its own. This tyrosine kinase activity is provided by the recruitment of a protein called JAK (janis-kinase) .
The receptor is activated, the JAK is recruited and there is dimerisation, then
this JAK gives the tyrosine kinase activity and then causes phosphorylation of the adjacent chain.
This phosphorylation of tyrosines then provides a site to which other proteins can bring (be recruited), one of these proteins is called STAT (signal transducer and activator of transcription). STAT is then dimerised (stabilising it) and then transported to the nucleus where it causes regulation of various genes via transcription.

Question 25

What does protein JAK do?

Answer 25

Provides tyrosine kinase activity to phosphorylate adjacent chain in growth hormone receptor

Question 26

Describe SMAD dependent signalling pathway activated by TGF-B

Answer 26

• Slightly different take on tyrosine kinase receptor
• It has serine threonine kinase activity
• Process is the same with dimerisation and autophosphorylation of specific sites acting as docking sites for other proteins
• SMAD - family of proteins that bind to receptor after it is activated which allow more interaction within the SMAD family. They will then migrate to the nucleus and regulate transcription

Question 27

Name three different signal transduction pathways

Answer 27

1. cAMP
2. Inositol P
3. Enzyme linked

Question 28

What signal transduction pathway do tyrosine kinase receptors use?

Answer 28

Enzyme linked

Question 29

What is the cAMP signalling pathway?

Answer 29

• Conformational change in G protein
• Effector: Adenyl Cyclase
• Inactivation: GTP phosphodiesterases

Question 30

What is the Inositol P signalling pathway?

Answer 30

• Conformational change in G protein
• Effector: Phospholipase C
• Inactivation: GTP phosphorylation

Question 31

What is the enzyme linked phosphorylase pathway?

Answer 31

• Conformational change and phosphorylation
• Effector: Proteins with SH2 domains
• Inactivation: Phosphatase

Question 32

Do pathways act in isolation?

Answer 32

No
A cell is constantly being bombarded with factors which may be conflicting, activation of receptors leading to opposite effects.
So the cell has to take all this information and do what is being promoted overall.
What the end result of the bombardment is depends on the interaction of the different signalling pathways and they modulate each others activity.