Intracellular Signalling - Signalling Pathway Components

Question 1

What can G-proteins bind?

Answer 1

GDP or GTP

Question 2

What is GTP?

Answer 2

High energy molecule that activates G-proteins

Question 3

What is function of GTPases?

Answer 3

Hydrolyse GTP into GDP (GDP switches G-protein off)

Question 4

What is prenylation?

Answer 4

Attachment of lipid-anchored membrane proteins via lipid tails

Question 5

How are G-proteins switched on?

Answer 5

Active when bound to GTP

1. Binding of 1st messenger to receptor induces conformational change in receptor that allows G-protein to bind to receptor
2. Stimulates G-protein to exchange its GDP for GTP
3. Switches it ‘on’

Question 6

How are G-proteins switched off?

Answer 6

1. G-protein hydrolyses GTP back to GDP

2. Inactive when bound to GDP

Question 7

What are the different alpha subunits of G-proteins?

Answer 7

1. Gi (inhibitory)
2. Gs (stimulatory)
3. Gg
4. G12,13

Question 8

What are the different alpha subunits of G-proteins coupled to?

Answer 8

Different effector enzymes

Question 9

What is effect when drug is coupled to activation of Gs?

Answer 9

Stimulates adenylate cyclase –> elevates cAMP

Question 10

What is effect when drug is coupled to activation of Gi?

Answer 10

Inhibits adenylate cyclase –> reduces cAMP level

Question 11

What are effects enzymes associated with:

1. Gs
2. Gi
3. Gg

Answer 11

1. Adenylate cyclase (stimualtion)
2. Adenylate cyclase (inhibition)
3. Phospholipase C (stimulation)

Question 12

What does GTP-bound Gs-alpha subunit bind to?

Answer 12

Binds to and activates adenylyl cyclase which catalyses conversation of ATP to cyclic AMP

Question 13

What is cyclic AMP broken to to and by?

Answer 13

Broken down to AMP by phosphodiesterases

Question 14

What is effect of Gg?

Answer 14

Increases DAG and IP3 levels

Question 15

How does cholera toxin affect Gs?

Answer 15

Affects Gs

1. Prevents GTPase activity of Gs so GTP remains bound to Gs so it stays switched ‘on’
2. This leads to over-stimulation of adenylate cyclase and accumulation of cyclic AMP
3. Elevated cAMP in intestinal epithelial cells increases loss of Cl- ions through chloride channels
4. Resultant osmotic gradient leads to water being excreted into intestinal lumen –> diarrhoea and dehydration

Question 16

What is pertussis toxin?

Answer 16

Whooping cough (affects Gi)

Question 17

What are effects of pertussis toxin on Gi?

Answer 17

1. Inhibits Gi –> prevents GDP/GTP exchange by Gi and Gi protein locked in ‘off’
2. Unable to inhibit adenylate cyclase
3. Accumulation of cyclic AMP
4. Increased insulin secretion and increased sensitivity to histamine

Question 18

Describe Gs before activation

Answer 18

Alpha subunit of Gs is bound to GDP, in ‘off’ state

Adenyl cyclase is inactive as hasn’t been activated by G-protein

Question 19

What is adenyl cyclase?

Answer 19

An enzyme with key regulatory roles in essentially all cells

Question 20

What happens when ligand binds to Gs receptor?

Answer 20

1. Causes G-protein to release GDP and swap it for GTP
2. Switches G-protein to ‘on’ state
3. GTP bound alpha subunit dissociates from the beta and gamma subunits

Question 21

What can GTP-bound Gs alpha subunit bind to and activate?

Answer 21

Adenyl cyclase –> catalyses conversion of ATP to 2nd messenger cAMP

Question 22

How is GTP hydrolysed back to GDP? What then happens?

Answer 22

The GTPase activity of the Gs-alpha subunit hydrolyses GTP to GDP (with release of inorganic phosphate, Pi), thus reverting the G-protein back to the “OFF” state

• The GDP-bound alpha subunit re-associates with the beta and gamma subunits
• Cyclic AMP is broken down to AMP by phosphodiesterases

Question 23

What is effect of Gs stimulation on cAMP?

Answer 23

Stimulates adenyl cyclase and increases cAMP

Question 24

What is effect of Gi on cAMP?

Answer 24

Inhibits adenyl cyclase and decreases cAMP

Question 25

Describe the opposing effects of Gs and Gi on cAMP levels

Answer 25

• A drug coupled to activation of Gs stimulates adenylate cyclase and elevates cAMP
• In contrast, a drug that is coupled to activation of Gi inhibits adenylate cyclase and reduces cAMP levels