lecture 20 - G protein coupled receptors and cAMP signalling

Question 1

ON mechanism - cAMP production

Answer 1

cAMP production is via the enzyme adenyl cyclase
there are 2 types of adenyl cyclase
type 1. in the plasma membrane composed of 2 TMDs and 2 catalytic domains (C1 and C2)
in the off state the catalytic domains are separated and together when activated and ATP can be converted into cAMP and pyrophosphate
enzyme is in off state unless its activated and bound to by G alpha s
type 2. soluble enzyme exists in the cytosol activated by increased HCO and Ca

Question 2

plant alkaloid activates the enzyme

Answer 2

forskolin

Question 3

some are regulated by calcium if u have isoform

Answer 3

AC8

Question 4

ON mechanism

Answer 4

adenylyl cyclase

Question 5

OFF mechanism - cAMP breakdown

Answer 5

Phosphodiesterases (PDEs)
11 isoforms - 8 types breakdown cAMP
others breakdown cGMP
PDEs shape the cAMP response can affect duration of the rise in cAMP, the amplitude (how high the levels go) and spatial localisation
caffeine inhibits PDEs so raises cAMP levels

Question 6

cilastazol

Answer 6

PDE3 inhibitor used for peripheral vascular disease (cAMP - vasodilation to improve blood flow)

Question 7

milrinone

Answer 7

PDE3 inhibitor used for failing hearts (cAMP - relaxes airway smooth muscle to reduce obstruction)

Question 8

roflumilast

Answer 8

PDE4 selective inhibitor used for COPD (cAMP - relaxes airway smooth muscle to reduce obstruction)

Question 9

sildenafil

Answer 9

PDE5 inhibitor used for pulmonary hypertension and erectile dysfunction (cGMP - reduces resistance and increases/sustains blood flow)

Question 10

OFF mechanism (2) inhibit cAMP production

Answer 10

some GPCR agonist activate the inhibitory G-protein, G alpha i which reduces activity (opposes G alpha s)

Question 11

OFF mechanism (3) cAMP removal

Answer 11

range of plasma membrane ABC-type/MRP transporters that can pump cAMP out the cell

Question 12

dynamics of cAMP

Answer 12

• different agonists increase cAMP levels but produce different responses in the same cells
• some physiological agonists produce cAMP - dependent responses but do no appear to change cAMP levels (we dont know where its changing and the changes in cAMP must be highly localised)

Question 13

how can cAMP be localised

Answer 13

restrict diffusion from plasma membrane to cytosol
target PKA to distinct sites and substrates in cells have GPCRs localised to different regions of the cell

Question 14

adenosine is a potent CFTR agonist

Question 15

A Kinase Anchoring Proteins - AKAPs

Answer 15

bind to PKA, and the AKAP goes to selective regions of the cell and take the PKA
AKAPs assemble signalsomes and brings all components together

Question 16

2 types of PKA

Answer 16

type I - cataytic unit has to go and find the substrate
type II is different as it can bind to AKAPs
it has a docking domain which it can bind to
the substrate is brought close to the catalytic subunits in type II by the AKAP

Question 17

HT31 peptide disrupts PKA-AKAP interaction

Question 18

ezrin (AKAP)targets PKA to CFTR but this requires scaffold protein NHERF1

Answer 18

NHERF1 has a PDZ binding domain that CFTR binds to and a ERM domain that ezrin binds to