lecture 21 Flashcards
what is the ON mechanism for cAMP production?
-cAMP made by adenyl cyclase (AC)
-protein-protein interaction between alpha s and AC = conformational change in AC (2 catalytic subunits come together)
when is adenyl cyclase off?
when alpha s isn’t bound
what is type 1 adenyl cyclase?
-found in plasma membrane (TMDs and catalytic domains)
-activated by forskolin (plant alkaloid that bypasses GPCR)
-9 isoforms (all activated by G alpha s)-binding causes C1/C2 domains to interact allowing production of cAMP from ATP
what does forskolin do?
-bypasses GPCR
-directly induces the conformational change of AC
what is AC 8 activated by?
increase in cytosolic calcium (could lead to increase in cAMP)
eg. in the airways
what is type 2 adenyl cyclase?
-cytosolic enzyme (AC10)
how is AC10 activated?
by increases in HCO3- (bicarbonate) and Ca2+
what are the OFF mechanisms?
- inhibit cAMP production
- breakdown cAMP (by phosphodiesterase)
- remove cAMP from cell
how is cAMP production inhibited?
-G alpha i reduces AC activity
-opposes stimulation by G alpha s
-lowers cAMP levels
how is cAMP broken down?
-phosphodiesterases
-11 isoforms but 8 breakdown cAMP
-expression is tissue specific
-degrade enzyme by breaking the phosphodiester bond
how are phosphodiesters important in ‘shaping’ the local cAMP signal?
it affects the:
duration
amplitude
spatial localisation
what inhibits PDEs?
caffeine
how are PDE inhibitors used?
clinically
what are the 3 PDE inhibitors that raise cAMP inside cells?
cilastazol
milrinone
roflumilast
what does cilastazol do?
-PDE3 inhibitor
-used for peripheral vascular disease
-cAMP causes vasodilation = improved blood flow
what does milrinone do?
-PDE3 inhibitor
-used for failing hearts
-cAMP increases heart rate and inotropy
what does roflumilast do?
-PDE4 selective inhibitor
-used of COPD (chronic obstructive pulmonary disease)
-cAMP relaxes airway smooth muscle = reduced obstruction
how is cAMP removed from cells?
-by ABC transporters that actively pump cAMP out the cell
what is the summary of the cAMP pathway?
- stimulatory agonists working through alpha s - impact plasma membrane form of adenyl cyclase to make cAMP which activates protein kinase A
- inhibitory agonists (alpha i) that block release of cAMP from adenyl cyclase
what is the cAMP pathway?
- cAMP acts through cAMP-dependent protein kinase A (PKA)
- this catalyses transfer of terminal phosphate of ATP to serene or threonine residues on selected proteins
- this phosphorylation can influence either the localisation or activity of the substrate
how is cAMP released from stimulatory agonsists and where does it go?
- by GPCRs where alpha subunit is bound to adenylyl cyclase on plasma Membrane surface
- soluble adenylyl cyclase activates cAMP and goes to the ABCC4 transporter
what inhibits stimulatory agonists?
cholera toxin
what does EPAC do?
-binds to cAMP
-stimulates phospholipase C isoform (PLCe) and Rap1
-this releases DAG and IP3
what are the problems with the linear pathway?
- different agonists increase cAMP levels but produce DIFFERENT responses in SAME cell
- some physiological agonists produce cAMP dependent responses but don’t change global cAMP levels
what do these findings suggest?
- changes in cAMP must be highly localised (compartmentalised) to spatially distinct areas inside cells
-changes in cAMP likely to be agonist-specific
how can cAMP signalling be compartmentalised?
1.have GPCRs localised to different regions of the cell
2. restrict diffusion of cAMP from pm to cytosol
3. target PKA to distinct sites and substrates in cells
what are some examples of cAMP being compartmentalised?
-CFTR activity in epithelial cells
-isolated heart cell response to different cAMP agonists
how do PDEs restrict diffusion?
what is patch clamp?
an experimental method to study how ions flowing through channels can change membrane potential.
what are the 4 types of patch clamp configuration?
outside-out, cell attached, whole cell, inside-out
what is the cell attached patch experiment?
-adenosine added to bath in exp 1 and pipette in exp 2
- in exp 1 = not much happens-channel cant be activated, forskolin added to bath soon and activates all AC - big increase in cAMP in cell
- in exp 2 = makes cAMP and activates some CFTR - antagonist added and shows less activity
what is the cell attached configuration?
tight contact created between pipette and plasma membrane
what does inhibiting PDE4 do?
-eliminates compartmentalised cAMP signalling in airway cells
how do MRP transporters alter cAMP levels?
-involved in regulating cAMP deponent processes in epithelial cells
eg. CFTR and intestinal Cl- secretion
what occurs in intestinal Cl- secretion with MRP4?
-monolayer of intestinal cells put into Ussing chamber to measure short circuit current
-cAMP = membrane permeable analogue
-glybenclamide = blocker of CFTR
how can cAMP signalling be compartmentalised?
-target PKA (effector) to distinct sites in cells
-happens through A kinase anchoring proteins (AKAPs)
what is the function of cAMP?
-passes info down signalling pathway activating cAMP dependent protein kinase (PKA)
-cAMP binding to PKA induces conformational change = release and activation of catalytic subunits
what are AKAPs?
- PKA2 binds to AKAP via PKA docking domain on Rll
-R submits bind cAMP but just change confirmation and can phosphorylate substrates nearby
-AKAPs have own targeting domain - brings PKA close to substrates
-help assemble signalling complexes to form signalling hubs or signalsomes
what is the role of AKAPs in activation of CFTR?
-from inside out patch
-ATP alone doesn’t activate channels
-PKA plus ATP = channel activation
-if PKA present via AKAP it should be able to:
1. activate CFTR by cAMP alone (+ATP)
2. block activation by cAMP by preventing PKAll binding to AKAP
what is HT31P?
-control peptide
-doesnt affect PKAll-AKAP interaction
what is HT31?
-short peptide
-disrupts PKAll-AKAP interaction
what is the conclusion of the experiment?
-PKA must be anchored very close to CFTR in the excised patch and must be involve an AKAP
-control did nothing
what study was done by Ezrin (AKAP) targeting PKA to CFTR?
-Ezrin targets PKA to CFTR
-binds via NHERF1
what are the properties of NHERF1?
- has binding domain for CFTR
- ERM domain that binds to Ezrin
what does activation of CFTR require?
AKAP, ezrin (linked to CFTR by scaffold protein), NHERF1
