CBS - Intracellular Signalling

Question 1

List some common cellular responses (and examples of each).

Answer 1

Change in metabolic activities:
- glucagon switches liver from synthesising glycogen to breaking down glycogen

Secrete and release:
- binding of antigen to mast cell stimulates the secretion of histamine

Changes in gene expression:
- Epidermal Growth Factor (EGF) activating genes involved in cell growth

Sensory perception:
- light activation of rhodopsin

Question 2

What factors act as extracellular signals?

Answer 2

Amino acids (and derivatives):

• glutamate
• adrenaline
• dopamine, etc.

Steroids:

• oestradiol
• testosterone
• cortisol
• aldosterone, etc.

Prostaglandins (eicosanoids) – derived from arachidonic acid

Proteins and peptides:

• insulin
• glucagon
• growth factor
• EGF, etc.

Gases

• NO
• CO

Question 3

List the different ways for cells to signal to each other.

Answer 3

ENDOCRINE:
Signal produced by cells in one part of body and travels in blood to target cells somewhere else

AUTOCRINE:
Signal acts on the same cell that produces it

PARACRINE:
Signal produced by cell and acts on other cells that are very close

CONTACT DEPENDENT:
Signal is integral part of one cell and interacts directly with another cell

NEURONAL:
Electrical signal transmitted down cell and message to another via synapse

Question 4

Why is the receptor important in communication between cells?

Answer 4

The cell must express the receptor in order to respond to the signal.

The receptors have high selectivity and high affinity.

The signal can bind to different types of receptors:

• β adrenergic receptor (adrenaline)
• α adrenergic receptor (adrenaline)

The signal is eventually turned off.

Question 5

Where are the different locations that receptors can be, and how do they work?

Answer 5

1. Cell surface receptor
   - the hormone is hydrophilic e.g. adrenaline
   - the binding of the hormone triggers a response inside the cell
   - the hormone does not “enter” the cell
2. Intracellular receptor
   - the hormone is hydrophobic e.g. steroid hormones
   - the hormone crosses the plasma membrane
   - the hormone binds to the receptor in the cytosol and triggers a response inside cell

Question 6

What are the different types of signalling?

Answer 6

Through the binding of the signal to the receptor, we can get:
- depolarisation of membrane due to flow of ions – (acetylcholine)

• direct activation of transcription factor
  steroid
• generation of secondary message inside cell (glucagon – cAMP)
• direct activation of enzymatic kinase cascade (EGF – MAP kinase pathway)

Question 7

Describe the direct activation of transcription factors via steroid hormones.

Answer 7

Steroid hormones contain a hormone binding domain, a DNA binding domain and a domain for interacting with other transcription factors.

The binding of the steroid induces a conformational change that allows DNA binding and activation of transcription of target genes.

The sequence specific DNA binding domains are hormone response elements in sequence of target genes.

They are ligand–dependent transcription factors.

Question 8

Give some examples of secondary messages inside the cell.

Answer 8

• cyclic AMP
• IP3/DAG
• Ca2+
• nitric oxide
• cyclic GMP

Question 9

List the second messengers generated by GPCRs (G-protein-coupled receptors).

Answer 9

Activation of adenylyl cyclase can generate:
- cAMP

Activation of phospholipase C can generate:

• IP3
• DAG

Question 10

Describe how G-proteins are activated.

Answer 10

1. A signal (e.g. adrenaline) binds to the receptor.
2. The G-protein (GDP bound) associates with receptor.
3. GTP/GDP exchange occurs on G-protein (now GTP bound).
4. G-proteins dissociates into a (GTP bound) and b,g
   subunits
5. The a subunit (with GTP bound) activates effector enzyme.
6. The effector enzyme produces a 2nd messenger.
7. GTP hydrolysed to GDP, and G-protein complex re- associates, signalling ends.

Question 11

Describe cAMP dependent protein kinase A (PKA).

Answer 11

It is a tetrameric enzyme, with 2 regulatory (R) and 2 catalytic (C) subunits (R2 C2).

cAMP binds to the regulatory subunit and the tetramer dissociates.

The catalytic monomers (C) are now active enzymes. This produces cAMP dependent protein kinase (PKA).

Question 12

Describe the cAMP-mediated effects on glycogen breakdown.

Answer 12

cAMp activity encourages glycogen breakdown.

It encourages glycogen breakdown by activating phosphorylase kinase b, turning it into phosphorylase kinase a.

The phosphorylase kinase a catalyses the conversion of phosphorylase b to phosphorylase a (active).

The phosphorylase a then catalyses the conversion of glycogen to glucose-1-phosphate, ready to be used for further metabolism.

The cAMP also inactivated the enzymes used for creating glycogen, glycogen synthase a, phosphorylating it to create inactivate glycogen synthase b.

Question 13

What effect does cAMP have on gene transcription?

Answer 13

PKA phosphorylates CREB (cAMP response element binding protein).

CREB binds to specific sequences in target genes and stimulates transcription.

This allows for long term adaptation to starvation, which causes changes in gene expression.

Question 14

Describe the cellular cascade that results from activation of the Gq subunit.

Answer 14

Some GPCRs contain Gαq (Gq) subunits.

The dissociated Gq activates phospholipase C.

Phospholipase C cleaves inositol phospholipids in the membrane. Examples such as:

• diacylglycerol (DAG)
• inositol 1, 4, 5 trisphosphate (IP3)

IP3 activates Ca2+ channels in the endoplasmic reticulum.

The Ca2+ concentration increases in the cytosol, and the DAG, together with Ca2+, activates protein kinase.

e.g. α1-adrenergic (adrenaline) receptor

Question 15

Describe the direct activation of enzymatic kinase cascade.

Answer 15

The binding of EGF triggers the autophosphorylation of tyrosine residues in cytoplasmic domain of the Receptor Tyrosine Kinase (RTK).

The adaptor proteins contain phosphotyrosine binding domains:

• SH2 (src – homology 2)
• PTB (phosphotyrosine binding)

Adaptor proteins Grb2 and SOS bind to receptor. This complex activates the exchange GDP-Ras -> GTP- Ras

Question 16

Describe the Ras protein.

Answer 16

Ras is a monomeric G-protein.

GTP-Ras triggers a kinase cascade. MAPKKK (mitogen activated protein kinase kinase kinase) activates MAPKK that activates MAPK that activates transcription factor.

This is the Ras-MAPkinase pathway. There is no secondary messenger

Question 17

How do hormones functions overlap?

Answer 17

One hormone can combine to different receptors. So, receptors can trigger different pathways.

Convergence:
- different signals trigger different pathways but cause the same effect in the cell

Cross talk:

• different signals trigger different pathways that block each other
• EGF signalling via PTK and Ras-MAP kinase but adrenaline inhibiting one of the steps via the action of PKA.

Question 18

(from tutorial)

Explain what is meant by the term ‘second messenger’.

Answer 18

Second messengers are molecules that relay signals received at receptors on the cell surface

The activation of the cell-surface receptor by the ligand leads to rapid amplification of the intracellular molecules.

Question 19

Give two other examples of second messengers other than cAMP.

Answer 19

Cyclic GMP, inositol trisphosphate, diacylglycerol, and calcium.

Question 20

Describe the structure, biochemical properties and functional role of a typical Gs protein.

Answer 20

Heterotrimeric GTP-binding proteins (G-proteins) are composed of three subunits, α, β, and γ. In a resting state, G-protein exists as the GDP-bound α-subunit complexed with the β- and γ-subunits.

The α-subunit of the G-protein has GTP hydrolysing activity and slowly converts GTP to GDP.

When bound to GDP, the α-subunit dissociates from the adenylate cyclase, which then becomes inactive.

When the original signal dissociates from the receptor, the receptor undergoes a ‘conformational change ’ and all 3
subunits of the G-protein re-associate. i.e the signalling mechanism goes to the OFF position.

Question 21

How are signalling mechanisms involving second messengers such as cAMP switched off?

Answer 21

The concentration of cAMP in the cell is restored to its normal low level by action of phosphodiesterase.

The cAMP dependent protein kinase then ceases to function and the cellular cascade
system stops.

Question 22

In gut epithelial cells, raised levels of cAMP activate a cascade process, via a cAMP dependent protein kinase, that eventually leads to the phosphorylation of a transport protein which controls the secretion of a Cl-, Na+ and HCO3- -rich digestive fluid into the lumen of the gut.

What would be the biochemical consequences of the adenylate cyclase being permanently activated?

Answer 22

Diarrhoea, as a lot more water will be released from the gut as all the solutes are also leaving, so they take the water with them via osmosis. You can lose litres of water in a few hours.

This also facilitates the loss of electrolytes, which can be fatal.

Question 23

The urgent aspect of treatment of a patient with cholera is to replace the huge volume of water and salts lost through diarrhoea. Oral rehydration treatment (ORT) involves drinking a solution made with glucose and salt.

Explain how this would maintain Na+ transport into the cell (with water following the Na+ ions).

Answer 23

It works through the Na+-glucose symport in the human intestine. The movement is due to a combination of sodium chemical potential and membrane potential.

As the glucose if brought in, the sodium is brought inside the cell with it. Thus, the water follows the salt and is able to rehydrate the patient safely.

The cholera toxin will degrade eventually, in a few days. The ORT will help the cells survive until then, and then they can recover.

Additionally, the alpha Gs proteins that have been affected will also be degraded, and replaced with new proteins that function normally.

Question 24

11. As cholera is a disease of developing countries, explain why oral rehydration therapy is preferable to vaccination programmes, treatment with antibiotics, or intravenous rehydration with glucose and saline solutions.

Answer 24

The other options are more expensive in this scenario, and antibiotics may not work as bacteria may become resistant.

IV drips are also not easily available in this scenario. Vaccines haven’t proven very effective.