PSC2002/L21 GPCRs & cAMP Signalling

Question 1

How long does a surge in Calcium last?

Answer 1

Seconds

Question 2

Describe the ON mechanism for cAMP production.

Answer 2

2 major types of adenylyl cyclase (membrane-bound & soluble)
Requires active alpha-sg protein bound (inbuilt timing system to turn off)
Hydrolyses GTP and inactivates

Question 3

What can activate all/some forms of adenylyl cyclase?

Answer 3

Plant alkaloid forskolin (bypasses GPCR)
Some activated by cytosolic calcium

Question 4

What activates cytosolic AC 10? (2)

Answer 4

Increases in HCO3- & Ca2+

Question 5

Give 3 OFF mechanisms for cAMP.

Answer 5

Inhibit cAMP production
Breakdown cAMP
Remove cAMP from cell

Question 6

Describe inhibition of cAMP production. (4)

Answer 6

Some GPCR agonists activate inhibitory G-protein Galpha
Reduces AC activity
Opposes stimulation by Galpha
Lowers cAMP levels

Question 7

What causes whooping cough? (2)

Answer 7

Bordetella pertussis toxin switches off Gai
cAMP levels remain high

Question 8

How many isoforms of phosphodiesterases (PDEs) are there?

Answer 8

11
8 break down cAMP
Others break down cGMP
Some break down both

Question 9

Describe the action of phosphodiesterases.

Answer 9

Tissue-specific
Shapes local cAMP signal duration, amplitude and spatial localisation

Question 10

What inhibits PDEs?

Answer 10

Caffeine
Many PDE inhibitors used clinically to treat disease symptoms

Question 11

Give 2 examples of PDE3 inhibitors in clinical use.

Answer 11

Cilastazol - for peripheral vascular disease - increases cAMP for vasodilation +blood flow
Milrinone - for heart failure (increases HR & inotropy to improve heart function)

Question 12

Give a selective PDE4 inhibitor.

Answer 12

Roflumilast - for COPD
Increase cAMP, relaxes airway SM to reduce airway obstruction

Question 13

Describe the removal of cAMP from cells.

Answer 13

Range of ABC transporters pump cAMP actively from cell

Question 14

What can affect duration and amplitude of cAMP signals?

Answer 14

MRPs

Question 15

Give 2 problems with the linear cAMP pathway.

Answer 15

Different agonists increase cAMP but produce distinct responses in SAME cells
Some physiological agonists produce cAMP-dependent responses but don’t change global cAMP levels

Question 16

How are changes in cAMP localised?

Answer 16

Compartmentalised to spatially-distinct microdomains inside cells
Changes in cAMP are agonist-specific

Question 17

How can cAMP signalling be compartmentalised? (3)

Answer 17

GPCRs localised to different regions of cell
Restrict diffusion of cAMP from pm to cytosol
Target PKA to distinct sites & substrates in cells

Question 18

Describe how toe execute a cell-attached path experiment of CFTR activation by ADO in polarised airway cells.

Answer 18

Add ADO to pipette
Add ADO to bath
Two separate experiments

Question 19

How was it determined that PDE4 eliminates compartmentalised cAMP signalling in human airway cells?

Answer 19

Patch clamp experiment
ADO to bath +/- PDE inhibitor
By inhibiting PDE, stop compartmentalising cAMP stores

Question 20

What is the role of A(2B)R?

Answer 20

Interact with CFTR
Localised in nature
When added to bath, cAMP can bypass PDE4 and activate CFTR

Question 21

What is the role of AKAPs? (3)

Answer 21

Binds to type II PKA through docking domain
Can be targeted to pm, organelles and subcellular structures via targeting domain to bring PKA closer to substrates
Help assemble signalling complexes to form signalling hubs or signalsomes

Question 22

What should be possible if PKA is present in a complex with CFTR via an AKAP? (2)

Answer 22

Activate CFTR by cAMP alone (+ATP)
Block activation by cAMP by preventing PKAII binding to AKAP

Question 23

What is required by ezrin (AKAP) to target PKA to CFTR?

Answer 23

Adaptor protein NHERF1
Binds to last 4 aa on CFTR
NHERF1 has PDZ1 binding domain that CFTR binds to (DTRL motif) & ERM domain that binds Ezrin