Lecture 05 - Intra-cellular Signalling

Question 1

What are G-proteins?

Answer 1

1. These are guanine nucleotide binding regulatory proteins
2. They are transducing proteins
3. They mediate the signal from the receptor to the effector
4. There are many G-proteins but they all work in generally the same way

Question 2

What is the structure of a G-protein?

Answer 2

1. Heterotrimeric - they have 3 different subunits, alpha, beta and gamma
2. GTPases - present within the alpha subunit which hydrolyse GTP to GDP + Pi

Question 3

What is the basic mechanism of the G-protein?

Answer 3

1. One receptor can activate several G-proteins, leading to magnification of the signal
2. Step 1 - no hormone/NT bound thus receptor is inactive
3. GDP is bound by the alpha subunit in the non-signalling state
4. Hormone/NT binds to the receptor
5. Receptor interacts with and activates G-protein
6. GDP drops off allowing GTP to bind to the alpha subunit binding site
7. The binding of the GTP causes dissociation of the G-protein
8. The alpha subunit and the GTP dissociate and signal to the effector thus stimulating it
9. Beta and gamma subunits stay together, they can also signal
10. Once activated, the signalling system which activates the effector (G-protein) must be switched off, done within the alpha subunit
11. Contains a GTPase which hydrolysed GTP to GDP and Pi
12. This deactivates the alpha subunit
13. It now reassociates with the other units

Question 4

What are examples of G-proteins?

Answer 4

1. Gs

2. Gi

Question 5

How does Gs work?

Answer 5

1. This G-protein stimulates cAMP

2. It may be affected by the actions of the cholera toxin

Question 6

How does the cholera toxin work?

Answer 6

1. The toxin inhibits GTPase
2. Prevents the hydrolysis of GTP
3. Results in constant activation of the subunit and adenylyl cyclase
4. Elevates cAMP levels
5. Causes excess secretion of water in the GI tract
6. Leads to diarrhoea and possibly death if not treated

Question 7

How does Gi work?

Answer 7

1. This G-protein also stimulates cAMP

2. May be affected by the actions of the pertussis bacteria

Question 8

How does the pertussis toxin work?

Answer 8

1. Toxin blocks the activation of the alpha subunit
2. Prevents it from inhibiting adenylyl cyclase activity
3. Leads to a rise in cAMP levels

Question 9

How much does adenylyl cyclase work?

Answer 9

1. Is an enzyme that puts the phosphate in ATP in a cyclic arrangement
2. Causes it to be converted to cAMP, a second messenger
3. This may be deactivated by converting it to a linear phosphate (5 AMP)
4. This is done by cAMP phosphodiesterase (PDE)

Question 10

How does cAMP function?

Answer 10

1. The molecule acts as a second messenger
2. Activates protein kinase (PKA)
3. Causes the phosphorylation of specific target proteins

Question 11

How do the cAMP levels?

Answer 11

1. The level of cAMP can be raised by activating adenylyl cyclase
2. Raised by also inhibiting cAMP - PDE, which hydrolyse cAMP to 5’AMP

Question 12

How do Gq/G11 proteins work?

Answer 12

1. Class of G-protein coupled receptors which activate phospholipase C
2. Is an enzyme that acts as an effector
3. Activation causes the formation of 2 second messengers
4. These are known as DAG and InsP3

Question 13

What is the mechanism of Gq/G11 proteins?

Answer 13

1. A hormone binds to the receptor
2. Activates the G-protein which activates phospholipase C
3. Acts on a lipid in the membrane (PIP2, phosphatidylinositol 4,5-biphosphate)
4. This is hydrolysed into DAG (diachlglycerol) and InsP3 (inositol 1,4,5-triphosphate)

Question 14

How does DAG work?

Answer 14

1. DAG activates membrane bound protein kinase C
2. Causes phosphorylation
3. Leads to a cell response

Question 15

How does InsP3 work?

Answer 15

1. Causes the release of calcium ions from intracellular stores
2. Calcium causes protein phosohorylation
3. Leads to a cell response

Question 16

How does a receptor tyrosine kinase work?

Answer 16

1. Binding of the appropriate molecule causes activation

2. This causes protein phosphorylation by activating tyrosine kinase

Question 17

How does a protein kinase work?

Answer 17

1. Protein kinase add phosphate groups causing phosphorylation

Question 18

How does protein phosphatase work?

Answer 18

1. This removes phosphate groups causing dephosphorylation

2. Adding/removal of phosphate groups is a way of regulating protein/enzyme activity

Question 19

How does glycogen metabolism work?

Answer 19

1. cAMP dependent protein kinase (pKA) — glycogen synthase — glycogen synthesis
2. cAMP dependent protein kinase (pKA) — glycogen phosphorylase kinase — glycogen phosphorylase — glycogen breakdown
3. cAMP causes a cascade/amplification reaction at each stage

Question 20

What happens when cAMP have increased levels?

Answer 20

1. Activates glycogen breakdown

2. Inhibits glycogen synthesis

Question 21

What happens when the level of cAMP increases?

Answer 21

1. An increase in the level of cAMP causes the activation of cAMP dependent protein kinase causes:
2. Phosphorylation of glycogen synthase - this deactivates it to prevent glycogen synthesis
3. Phosphorylation of phosphoprotein phosphatase - deactivates it to prevent removal of phosphate groups
4. Phosphorylation of glycogen phosphorylase kinase - will cause phosphorylation of glycogen phosphorylase
5. Glycogen phosphorylase will cause hydrolysis of glycogen to glucose-1-phosphate

Question 22

What is the equation for glycogenesis?

Answer 22

1. Glycogen (n) + UDP Glucose —-> glycogen (n+1) + UDP

2. Carried out by glycogen synthase

Question 23

How is phosphoprotein phosphatase inactivated?

Answer 23

1. The activation of cAMP dependent protein kinase causes:
2. Phosphorylation of protein phosphatase inhibitor
3. Causes it’s activation
4. In turn this binds to the active phosphoprotein phosphatase
5. Causes the enzyme to be deactivated

Question 24

What happens when cAMP levels are decreased?

Answer 24

1. This inhibits glycogen breakdown

2. Activates glycogen synthesis

Question 25

What is the mechanism of decreased cAMP levels?

Answer 25

1. The activation of the inactive phosphoprotein phosphatase (due to low cAMP) causes: 2. Dephosphorylation of glycogen phosphorylase - inactivates it thus preventing glycogen breakdown 3. Dephosphorylation of glycogen phosphorylase kinase - inactivates it, preventing activation of glycogen phosphorylase 4. Dephosphorylation of glycogen synthase - activates if allowing for glucose to be converted into glycogen

Question 26

What is the equation of glycogenolysis?

Answer 26

1. Glycogen (n + 1) + Pi —> glycogen (n) + glucose-1-phosphate 2. Carried out by glycogen phosphorylase