cAMP signalling Flashcards
How cAMP signaling activated, what is cAMP, effector proteins, desensitisation
Cyclic AMP is a small molecule that serves as an intracellular second messenger of many hormones & neurotransmitters
Generate inside cells by the action of plasma membrane-adenylyl cyclase and quickly diffuses throughout cell (even through nucleus)
cAMP is detected by PKA effectors which phosphorylates target proteins EPAC, CNGCs, POPEYE and cAMP GEF
PDE is only family that degrades cAMP into AMP. Different localised degradation methods depending on type
cAMP production and degradation
Universal and ubiquitous signalling mechanism. 60 different Gs receptors expressed in different cell types with different physiological outcomes all produces cAMP
Gs coupled ligand bound receptor is activated so G alpha s is stimulated to bind and activate adenylyl cyclase to produces cAMP which binds to PKA and EPAC in cytoplasm.
PDE (‘sinks’) are highly specific and degrades cAMP in localised areas. If a receptor mediated event produces so much cAMP that PDE can’t cope, cAMP will increase in that area past the receptor threshold for activation
How is cAMP mediated signalling compartmentalised and examples
cAMP can have different physiological effects due to relationship between localisation of PDE, adenylyl cyclase and cAMP microdomains they reside in govern signalling.
Adenylyl cyclases (ACs) are localised to specific parts of the membrane (ex. lipid rafts) to produce cAMP locally.
Differential expression of PDEs for compartmentalisation. Ex. In nasal cavity of dogs, through staining specific proteins, saw AC3 (in membrane as expected), PDE4A in bottom of neurons so cAMP only affects protein that is likely near the top. PDE2 is not in nasal olfactory neurons (targeted to a different set of neurons)
AKAPs (A-kinase anchoring proteins) physically anchor PKA and other cAMP effectors to specific sites.
Selective expression of receptors (e.g., β1-adrenergic at the plasma membrane vs β2-adrenergic in T-tubules in heart cells) shapes where cAMP is made.
How was the compartmentalisation of cAMP confirmed
Previously had to destroy cells and probe with antibodies, destroying the compartmentalisation. Immunohistology did not provide sufficient quantitive information to visulasie cAMP gradients at high resolution.
With FRET probes can track activation of PKA a proxy for cAMP localisation
Use YFP (absorbs 480 nm to release 545nm) fused to catalytic PKA subunits and CFP (absorbs 430 nm to release 480 nm) fused to regulatory subunits and genetically encode the sensor in living cells in real-time
Saw distinct clouds of cAMP throughout cell
Ex. transfect sensor into heart cell
Under no beta-1 adrenergic stimulus no/low FRET
With stimulus went to distinct positions in cell- sarcomere (calcium stores) where cAMP activates quicker Ca release
First to link cAMP clouds/PKA localisation to physiological response
AFM used to see 3D position of T-tububles in cells and confirmed cAMP localisation there with FRET based sensor
Targets of the cAMP pathway for clinical applications
Wide variety of receptors that are able to regulate adenylyl cyclase activity (ex. GPCRs)
Multiple adenylyl cyclase isoforms (9/10)
Cell type specific expression of targets for PKA (EPAC, CNGC, POPEYE)
Isoforms of PKA, activation, differences
PKA phosphorylates 1500 proteins. Inhibitor drugs have detrimental effects as not specific enough
2 cAMP binds to regulatory subunit, disassociating complex, releasing catalytic subunits to phosphorylate target substrates
2 isoforms of regulatory subunit: PKA-RI (predominantly cytosolic to monitor gradients of cAMP in cells) and PKA-RII (anchored at specific intracellular sites by AKAPs)
Role of PKA catalytic subunit isoforms is unknown
AKAPs function, mechanism, use in clinic
A-kinase anchor proteins that sample cAMP gradients in cells. There are AKAPs for every major cell location (nucleus, FA, membrane, etc) to bind PKA to specific locations.
Some AKAPs can interact with other proteins such as PDEs, kinases, etc for faster spatiotemporal control of signalling
PKA-RII has a two alpha helix interface that the AKAP peptide binds with high affinity.
As a PKA inhibitor, can produce pseudo-substrate with the PKA consensus motif that AKAP will bind instead, causing improper localisation of PKA
EPAC: isoforms, difference, function,
EPAC has two isoforms (Epac-1; wildly expressed and Epac-2;), with Epac-2 having 2 cAMP binding domains
EPAC is autoinhibited at rest by the GEF domain blocked by the regulatory domain aand binds cAMP to activate (with a lower affinity than PKA)
EPAC is a GTP exchange factor (GEF) to mini-G-proteins Rap1 (stabilises VE-cadherin mediated cell-cell contacts, activates ERK, and induce SOCS-3) and Rap2
No small molecules that activate or inhibit EPAC successfully
cAMP as a vasorelaxant examples
Caffeine is a mild bronchodilator that inhibits PDEs, causing increase in cAMP, activating PKA, causing smooth muscle relaxation and opening airways
Salbutamol activates beta-2 receptor for bronchodilation in asthma
GPCR activaes AC producing cAMP that activates PKA activating EPAC.
EPAC activates ryanodine receptor via small GTPases (ex. Rap1) to release Ca sparks inducing membrane hyperpolarisation, reducing Ca influx. Decreases activity of smooth muscle voltage gated Ca channels, reducing Ca influx and promoting relaxation
Has opposing effects on Ca channels (activates NCX that pumps Ca out cell, VGCC inhibits bringing Ca in cell, causing net loss of Ca)
Ca binds fibres causing cell contraction
POPEYE: discovery, function, mechanism, structure
Incubated cell lysate with cAMP coated metal beads and did proteomics to identify proteins bound
Discovered protein POPDC with a canonical cAMP binding site. Has 3 membrane binding domains
Theory of how it functions: POPDC binds ion channels to activate. Binding cAMP removes it from ion channels stopping activation.
High expressed in cardiomyocytes and involved in pacemaking. Mutation causes cardiac arrhythmia and muscular dystrophy in patients
PDEology: types, targetting, differences
Cyclic nucleotides require large diversity of PDEs
11 different families and >100 different isoforms from these families arising from being encoded by multiple genes and alternative mRNA splicing
Active sites (catalytic region of 360 aa) are almost identical so need a way to target PDE targeting drugs for use in clinic (ex. viagra is a PDE5 encoded by only one gene so easy to target without many off-targets)
Differ in:
Ability to hydrolyse cAMP and cGMP (Vmax and Km values)
Regulatory properties
Intracellular localisation
Signalling complexes recruited to
PDE activation effect and clinical examples
Short term: PKA phosphorylates and activates PDE3, 4 and 8, reducing cAMP
Long term: PKA phosphorylates CREB inducing upregulation of PDE4 expression
PDE inhibitors induce the long term pathway, massively upregulating PDEs and making the drug less effective
PDE4 in clinic:
PDE4 inhibitors as antidepressants, cognitive enhancers, anti-fibrotic agents, anti-cancer and anti-inflammatory.
Ex. Anti-inflammatory: PKA activates CREB which increases transcription of IL-10 cytokine. PDE4 is widely expressed in inflammatory cell types and PDE4 inhibitors negatively modulates production of pro-inflammatory mediators at the level of mRNA expression.
Stimulate PDE4 in polycystic disease and prostate cancer
PDE4: isoforms, structure
4 isoforms: long (two UCRs), short (one UCR), super-short (part of a UCR), and dead-short (catalytically inactive)
Diversity in activation mechanism
Each has a conserved catalytic subunit (differs enough for diversity in targeting)
Regulatory domains (UCRs): autoinhibits catalytic domain
C-terminal region: specific and typically involved in substrate interactions
N terminal region: specific postcode sequence to target the PDE to cell compartment
Activation of each PDE4 isoform
Long form:
Low basal activity
PKA activation phosphorylates PDE4’s PKA consensus site (RRESW) in UCR1, causing PDE4 activation (opens up the transcap by weakening UCR1 and UCR2 interaction) via disassociation of regulatory domain
ERK phosphorylation decreases activity
Autoinhibition: Exists as dimer in which UCR2 transcaps the active site on the other monomer. UCR1 blocks where cAMP binds so not as active as short-form
Sustained activation: C terminal cis cap forms in phosphorylation stabilising the open conformation and preventing UCR2 from re-closing over active site
Short form:
Maintain basal activity when PKA is active since don’t have UCR1
ERK phosphorylation increases activity
Monomer
More active than long form
Super short form:
ERK phosphorylation decreases activity
Dead-short form: catalytically inactive due to severe N and C terminal truncation
ERK can phosphorylate PDE4 on catalytic subunit on ERK consensus site (P-X-S/T-P) which has nearby MAPK docking domain to allow it to bind
Some PDE4As doesn’t have ERK consensus site so can’t be phosphorylated
Activating PDEs in clinic: method, use,
Useful in polycystic disease and prostate cancer
Small molecule that binds UCR1 domain in PDE4 long isoform (specifically the PKA consensus sequence) to activate
PDE4 competitive inhibitors
Roflumilast to treat chronic inflammation in COPD. Very bad side effects so only use in very severe cases
In COPD, cAMP drives anti-inflammatory responses (increase of PKA activation phosphorylates Csk which inhibits LPS (inducer of inflammatory responses; TNF-alpha, ILs)) so increasing levels with PDE4 inhibition can be used.
T cells treated with LPS has large increase in PDE4 (especially the short form) to inactivate
Nausea (associated with PDE4D activation), headache and diarrhea (high affinity rolipram binding site (HARBS) is associated with gastric acid production)
Due to mode of action and lack of sensitivity for isoforms/subfamilies, since they are important in inflammatory and non-inflammatory functions
Apremilast is highly selective for PDE4s but no selectivity within the family.
Drug to treat psoriasis- can cause complete remission (highly successful)
Less side affects since poor brain transmission and low binding to HARBS
Better therapeutic index but higher dose = more severe symptoms
Competitive for cAMP
SUMOylation: definition, function
Small protein that adds to consensus site (Hyd-K-X-E) to change protein activity or localisation
PDE PKA phosphorylation with SUMOylation superactivates by locking in open conformation and protects against ERK phosphorylation
All long-forms (but 4A) can be SUMOylated
PROTAC: mechanism, example, advantages, disadvantages
Drug that doesn’t depend on active site inhibition
Recruits ubiquitin to POI to degrade- takes longer to work than direct inhibitor binding
Fused 4B specific compound to linker and E3 ligase.
KTX207 is extremely successful degrading shortform PDEs (dimerisation stops linker from recruiting E3 ligase)
Advantages:
Selectivity (short vs longforms) via warhead and linker design
Degrades protein ablating enzymatic and non-enzymatic functions (ex. some PDEs may act as scaffolds)
Enzymatic mechanism works at low doses as systematic degradation (less side effects)
Very long lasting as once inside cell PROTAC keeps working and is not consumed
No rescue from upregulation of target protein
Disadvantages:
Low bioavailability
Low BBB penetration for CNS
Has to recruit E3 ligase in cell and if not well expressed PROTAC will not work
Hook effect- concentration has to be in sweet spot
Ideal PDE4 inhibitior
High affinity, PDE4 specific
Isoform specific failing that sub-family specific
If not, non-brain penetrating
Good HARBS/LARBS profile
No effect on other active proteins (ex. kinases)
Good bioavailability
