L21 G-protein coupled receptors and cAMP signalling

What is the ‘ON’ mechanism for Type 1 adenylyl cyclase

• activated by binding of Galpha_S

• 9 isoforms all activated by Galpha_S

• Forskolin directly activates AC (By-passes need for GPCR)

• some ACs are also activated by increases in cytosolic Ca2+

what is forskolin

plant alkaloid

give an example of an AC activated by increases in cytosolic Ca2+

AC 8 -airways

What is the ‘ON’ mechanism for Type 2 adenylyl cyclase

• cytosolic enzyme (AC 10)

• Activated by HCO3- and Ca2+

what do OFF mechanisms do

• inhibit cAMP production

• breakdown cAMP

• remove cAMP from cell

how do OFF mechanisms inhibit cAMP production

some GPCR agonists activate Galpha_i which reduces AC activity, opposing stimulation by Galpha_s, lowering cAMP levels

how do OFF mechanisms breakdown cAMP using phosphodiesterases (PDEs)

• 11 isoforms (PDE1-11)

• 8 breakdown cAMP, others breakdown cGMP and some breakdown both cAMP and cGMP

• Expression is tissue-specific

• important in ‘shaping’ the local cAMP signal duration, amplitude and spatial localisation

• PDEs are inhibited by caffeine

• PDE inhibitors are used to treat disease symptoms

describe the clinical use of PDE inhibitors

increase cAMP inside cells to help reduce the negative ‘symptoms’ of some diseases

what is PDE’s role in cilastazol

PDE3 inhibitor used for peripheral vascular disease

• increase in cAMP- causes vasodilation to increase blood flow

what is PDE’s role in milrinone

PDE3 inhibitor used for failing hearts

• increase in cAMP-increases heart rate and inotropy to improve heart function

what is PDE’s role in roflumilast

PDE4 selective inhibitor used for COPD

• increase in cAMP- relaxes airway smooth muscle to reduce airway obstruction

what is cAMP signalling not defective in

• HF

• COPD

how do OFF mechanisms remove cAMP from the cell

using a range of plasma membrane ABC transporters that actively pump cAMP out of the cell (e.g. MRP4)

what do MRPs affect when removing cAMP from the cell in OFF mechanisms

duration and amplitude of the cAMP signal

• may also impact spatial aspects

what are problems with the linear pathway (dynamics) of cAMP signalling

• different GPCR agonists that increase cAMP produce distinct ‘responses’ in the same cells

• some ‘physiological’ agonists produce cAMP-dependent ‘responses’ but don’t change global cAMP levels

what does different GPCR agonists that increase cAMP produce distinct ‘responses’ in the same cells suggest

changes in cAMP must be highly localised (compartmentalised) to spatially-distinct areas inside cells (microdomains)

what does some ‘physiological’ agonists produce cAMP-dependent ‘responses’ but don’t change global cAMP levels suggest

changes in cAMP are likely to be agonist-specific

what do we need to fully understand cAMP signalling

have knowledge about both temporal and spatial changes in cAMP

how can cAMP signalling be compartmentalised

• have GPCRs localised to different regions of the cell

• Restrict diffusion of cAMP away from the pm to the cytosol

• Target PKA type II to distinct sites and substrates in cells

what is the experimental evidence for cAMP signalling to be compartmentalised methods

• CFTR activity in epithelial cells

• isolated heart cell response to different cAMP agonists

how do we target PKA type II to distinct sites and substrates in cells

role of A kinase anchoring proteins (AKAPs)

what can AKAPs be targeted to the pm and organelles via

the targeting domain

PKA type II binds to the AKAP via…

the PKA docking domain on RII

what does targeting AKAPs to the pm and organelles do

brings PKA type II close to its substrate

AKAPs help assemble signalling complexes to form…

signalling hubs or signalsomes

if PKA is present in a complex with CFTR at the pm, via an AKAP, (CFTR activation by PKR) then it should be possible to…

• activate CFTR by cAMP alone (+ATP)

• block activation of CFTR by preventing PKAII binding to the AKAP

what is HT31’s role as an AKAP in CFTR activation by PKA

a short peptide

• disrupts PKAII - AKAP interaction

what is HT31P’s role as an AKAP in CFTR activation by PKA

control peptide

• does not affect PKAII-AKAP interaction

PKA must be anchored very close to CFTR in the excised patch, and this must involve an…

AKAP

what does Ezrin (AKAP) target

PKA to CFTR

what does Ezrin (AKAP) targeting PKA to CFTR require

the adaptor protein NHERF1

describe the PDZ1 binding domain of NHERF1

CFTR binds to (DTRL motif)

describe the ERM domain of NHERF1

binds Ezrin

where does Ezrin function

human bronchial epithelial cells

other work provided experimental evidence that PDEs were also found to be active close to…

CFTR in the apical pm

the PDEs prevented diffusion of cAMP away from the pm into the cytosol, localising…

cAMP to the subapical compartment

what did the localisation of cAMP to the subapical compartment do

increase the efficiency of CFTR activation by GPCR agonists such as adenosine