GPCRs 2

Question 1

Describe desensitisation

Answer 1

1. GRK (6 members of family)
2. Phosphorylate agonist-bound receptor, when G-protein is not bound
3. Serine/threonine kinase
4. Enhances affinity of arrestin to bind to receptor- acts as plug to prevent G-protein from recombining and carrying on signalling

Question 2

What happens to electrical current when Gi/go receptors are activated

Answer 2

1. When Gi/go receptor activated potassium ions leave cell as beta subunit binds to channel
2. Cause increase in electrical current…

Question 3

What are 3 Mu-opiod receptors agonists

Answer 3

1. Morphine – partial agonist – no receptor reserve
2. Met-enkephalin – full agonist
3. DAMGO – full agonist
4. All have similar affinities, so potency is to do with efficacy

Question 4

What happens when to a full agonist activity when a receptors desensitise and give example

Answer 4

1. Effect of full agonists declines- desensitises
2. DAMGO has highest efficacy and also declined the most
3. Agonist with high efficacy is better at activating receptor so therefore more likely to undergo GRK and arrestin mediated desensitisation
4. Within a minute 99% of receptors were desensitised as so efficacious

Question 5

What happens when to a partial agonists activity when a receptors desensitise and give example

Answer 5

1. Morphine (partial agonist)- no decline
2. No receptor reserve
3. If something happens to cell that causes receptor level to decrease then morphine response will inevitably decrease
4. Whereas DAMGO has high receptor reserve so doesn’t need to activate many receptors

Question 6

Why doesn’t morphine desensitise

Answer 6

1. Activating PKC enhances morphine-induced desensitization – Introducing functional selectivity
2. But PKC has no effect on DAMGO
3. GRK mediates DAMGO-induced but not morphine induced desensitisation - so doesn’t regularly desensitise
4. DAMGO, but not morphine, causes arrestin translocation in HEK cells

Question 7

What is functional selectivity

Answer 7

1. Functional selectivity is the ability of a receptor to couple to more than one signal transduction pathway.
2. Different agonists acting at the same receptor have different actions

Question 8

Give example of functional selectivity in desensitisation

Answer 8

1. DAMGO activation induces sensitisation through classical route of GRK and arrestin
2. Morphine desensitises through PKC- probably phosphorylating steps
3. Same receptor in same cells but 2 different agonists can cause 2 different mechanisms of desensitisation
4. Inhibiting desensitisation may be easier if agonist doesn’t use GRK mechanism e.g. morphine

Question 9

Before functional selectivity what was the model

Answer 9

1. The linear model
2. Drug + receptor –> drug receptor complex –> conformational change –> activated DR - if efficacy
3. How well it drives from D+R to DR= affinity
4. How well it drives DR–> DR* = Efficacy - good at stabilising conformational activated state of DR

Question 10

What is another word for functional selectivity

Answer 10

1. Biased agonism

Question 11

What is new theory

Answer 11

1. Different agonists stabilise different conformational changes that are already happening
2. GPCRs are constitutively active- signal by themselves- fluid and moving in and out of states all the time
3. In flexible lipid membrane
4. every now and again an agonist will move itself into an active conformational state and signal

Question 12

What does a high and low efficacy agonists do in new theory

Answer 12

1. Binds to the receptor and waits for it to change conformational active state and if it gets to active state the high efficacy agonist is good at stabilising it in its active state
2. Low efficacy- Not as good at stabilising receptor in active state, signals for less time

Question 13

What does an inverse agonists do in new theory

Answer 13

1. Ligands that stabilise receptor in inactive state
2. Inverse agonists existing led to this idea

Question 14

How does new theory explain how two different agonists can cause desensitization by different mechanisms?

Answer 14

1. Must be more than one active conformational state

Question 15

What effect can more than one conformational state

Answer 15

1. How agonists cause receptor desensitization
2. Arrestin-based signalling
3. The G-protein activated by an agonist
4. Could this lead to improved drug design?

Question 16

How does more than one conformational state affect how agonists cause receptor desensitization

Answer 16

1. m-opioid receptors: PKC/GRK
2. b2 receptors: PKA/GRK
3. Both receptors – some agonists desensitise through PKC or PKA and some through GRK

Question 17

How does more than one conformational state affect arrestin-based signalling

Answer 17

1. Idea that you can exploit functional selectivity to enhance or reduce arrestin-based signalling is exciting
2. Arrestin is also scaffolding protein in its own right
3. If GPCR activated with agonist, signal through g protein, then desensitisation and arrestin binds
4. Arrestin mediated signalling starts
5. Activates different signalling pathways than with G-protein
6. Activation of c-Jun kinase cascade
7. Activation of MAP kinase cascade

Question 18

How does more than one conformational state affect the G-protein activated by an agonist

Answer 18

1. Alpha2adrenoceptor
2. Gi/go receptor – inhibition of cAMP
3. cAMP decreases when Agonist A applied
4. Only allows coupling through Gi
5. agonist B inhibit then increase levels
6. good at allowing through Gi and then also Gs
7. Agonist C allows coupling through both Gi and Gs
8. ONLY been seen in recombinant/heterologous cell systems
9. Different agonists drive activation of different G proteins at receptor

Question 19

How does more than one conformational state lead to new approaches to drug design

Answer 19

1. Reduced side-effects
2. Different drugs good at stabilising different active states of a receptor
3. Some may give side effect and the others not
4. Cell-specific effects
5. EG. Gi/Go-coupled receptor, you want Gi effects, but MAPkinase activation by arrestin recruitment is a side-effect, so design a drug that activates Gi/Go, but doesn’t recruit arrestin