Second Messengers Flashcards
Give an overview of second messengers - cAMP?
GPCR is in the plasma membrane - interfacing with a ligand
This activates adenylyl cyclase - which generates a molecule catalytically (cAMP) - which will diffuse away from the site of generation (until it is degraded)
cAMP will interact with other molecules and can initiate changes
Adenylate cyclase is docked to the active subunit of the heterotrimeric g-protein - it is fully active - therefore generating cAMP at a given rate
Describe cyclic AMP?
Discovered in 1959 by Sutherland
Produced from ATP - using adenylyl cyclase, which removes 2 Pi groups
[cAMP] range 0.1 to ~1mM
It stimulates glucose mobilisation from glycogen, fight or flight response, triglyceride breakdown
Acts through a protein kinase (primarily)
It can be broken down by cyclic AMP phosphodiesterase - we attack the organic chemistry, but conserve materials (energy efficient)
Describe the production of cAMP?
- Ligand binding alters the conformation of the GPCR, exposing the binding site for Gs protein (alpha, beta and gamma subunits)
- Diffusion in the bilayer leads to association of ligand-receptor complex with Gs protein = greatly weakening the affinity of Gs for GDP
- GDP dissociates, allowing GTP to bind - causing the a subunit to dissociate from the Gs complex, exposing the binding site for adenylyl cyclase
- The a subunit binds to/activates the adenylyl cyclase - now producing many cAMP
- The dissociation of the ligand then returns the receptor to its original conformation
- Hydrolysis of GTP by the a subunit returns a to its original conformation = dissociation from adenylyl cyclase (now inactive) - and reassociate with beta and gamma (reform Gs)
Describe adenylyl cyclase?
Large enzyme - 12 transmembrane domains
Converts ATP into cAMP - with a turnover of 1000min-1
It is located in sarcolemma
There are a variety of isoforms - 6 members, 50-92% identity, differential regulation Ga/Gby/Calmodulin (CaM)
Describe the cAMP phosphodiesterase?
Large number of isoforms - around 30
Mr 58-125 kDa
It contains a catalytic core (conserved) and then regulatory/targeting features
The default position of the enzyme is active - needs to be kept in relatively low concentrations
The different isoforms are activated and inhibited by different ligands
Example - Class V is cGMP specific and inhibited by Viagra
Describe the fight/flight response?
This is an example from cardiac muscle
When this response is engaged adrenergic agonists bind to the beta adrenergic receptor causing an increase in cAMP = stimulate protein kinase A
This brings about large changes in contractile cells - as increase in cAMP means the kinetics of contraction/relaxation are accelerated meaning the degree of shortening is decreased
This allows more blood to be released from the heart and increased nutrients to areas of the body that need to react in this fight/flight response
Describe the need of control with cAMP?
Control features needed to limit cAMP production to a short/small pulse (in time/ concentration) Control systems are short-circuited by pathogens for example: Cholera toxin: ADP ribosylates Ga, inhibits GTPase activity and extends activation Bordetella pertussis (whooping cough) injects soluble adenyl cyclase into host
How is cAMP production limited?
Receptor optimised for inactivity - needs a ligand to bind/activate
Receptor internalised via endocytosis into the cell
Ga hydrolyses GTP
GAP activates GTPase activity
What are the modes of signalling by cAMP?
There are 3 methods of how the signal propagates further into the cell
cAMP-dependent protein kinase
EPac (Exchange protein directly activated by cAMP)
Ion channel modulation
Describe the subunits of cAMP-dependent protein kinase A?
Tetramer - 2 copies of regulatory and 2 copies of catalytic
Regulatory - binding sites of ligand (cAMP)
Once cAMP binds the complex collapses
The lone catalytic site is now active
It has an autoinhibitory domain within the regulatory site
Catalytic - brings the terminal phosphate close to the phosphorylatable residue on a substrate protein for phosphoryl transfer
Describe the structure of cAMP-dependent protein kinase A?
Globular protein with a cleft (active site)
ATP bound to N-terminal lobe
Protein substrate in large C-terminal lobe
Specific contacts between substrate and protein - means different molecules bind
Kinase enzyme is deterred by: T at phos. site acidic residues at P-1 K at P+2 or F at P+4 -3 RRxSxxxx +4 - typical consensus sequence
Give some examples of targets of PKA?
Individual protein targets are phosphorylated on particular Ser- or Thr- residues to alter the function of that protein:
The phosphate group is removed
Phospholamban - accelerates Ca2+ uptake into SR
RYR2 - increases Po of channel
Tnl - reduces Ca2+ sensitivity of contractile protein
How is the signal (PKA) encoded/decoded?
Comparmentation
Enzymes are located at key structures in cells via binding partners
AKAPs (A-kinase Anchoring Proteins) are proteins located on sub-cellular structures
They bind PKA and other signalling proteins - which can include PDE, other kinases and phosphatases
They locates signalling machinery close to substrate proteins
Properties - PKA holoenzyme, targeting domain and other signalling components
What doe AKAPs do?
They associate with EC coupling proteins
There are different signalling circuits for L-type Ca2+-channel, RYR2 and PLB
Due to different enzymes attached to the AKAPs close to these proteins
Independent control of target phosphorylation, despite all using cAMP
This allows the cell to send multiple messages through the same signalling molecule
Example of signalling in the heart:
b1-AR: substantial (400%) enhancement in contractility, plus cardiotoxicity (when chronic)
b2-AR: modest (25%) enhancement in contractility, plus cardioprotective
Describe the overuse of signals?
This drives maladaptation and disease
This is as a result of receptors coupling to multiple signalling pathways - showing the pathways aren’t necessarily linear
Can increase contractility and apoptosis e.g. CaMKII can cross both affects from oxidative stress and ischemia
Give an overview of Ca2+ as a second messenger?
Starts at the plasma membrane
A ligand binds to a coupled receptor
If GPCR -> phospholipase C generates IP3
IP3 is a ligand for a IP3 receptor, allowing Ca2+ to exit the intra cellular store into the cytoplasm
Describe IP3 and its role?
It is a inositol sugar - called 1,4,5-triphosphate (IP3) - a phosphorylated monosaccharide Derived from a minor class of phospholipid (phosphatidylinositides) - mainly found on the inner leaflet of the membrane, that get gradually phosphorylated
Roles:
Contraction in smooth muscle (contracts slower than striated - due to Ca2+ diffusion)
Fertilisation - prevents the egg accepting a second sperm
Aggregation of platelets (blood clotting)
Secretion (cells of adrenal cortex)
Describe how phospholipase C is activated?
2 major routes - they activate different isoforms of the enzyme
In both - PIP2 is cleaved into IP3 and DAG
- GPCR (7 helix receptor) with a heterotrimeric G protein
Ligands can be: acetylcholine, serotonin, 5-hydroxytryptamine or photons of light - (platelet derived) Growth factors
Describe GPCR signalling to phospholipase C?
Agonists: light, odour, peptides, 5-HT ACh
G proteins: Gq, G11, G14, G16
both a and bg subunits activate phospholipase C
Gaq - PLC-b1
Gbg - PLC-b2
GTPase activity of Ga enhanced upon binding to PLC (turning off the signal)
Describe growth factors signalling to phospholipase C?
Class of receptors that have an enzymatic function on the cytoplasmic surface
Single transmembrane domain with a large extracellular domain for ligand binding
- The ligand can bind to two receptors - bringing the receptors physically closer together
Receptors dimerise and autophosphorylate - via kinases and tyrosine kinases - SH2 domain of PLCg docks onto phosphoreceptor
- PLCg phosphorylated (Y771, Y783, Y1254) and activated
PKA/PKC can phosphorylate and inhibit PLCg
Describe the intracellular store of Ca2+?
Stored within an impermeable membrane
Enzymatic/energy required pumps to move Ca2+ against it’s concentration gradient into the store
Buffer in the store - enhances the capacity
A Ca2+ channel to release the Ca2+ from the intracellular store - IP3 ligand
Describe the IP3 receptor?
Ca2+ channel (with the IP3 receptor on it) in the intracellular Ca2+ store is in the endoplasmic reticulum
3 isoforms, 2701-2745 residues - they assemble as homotetramers
Most of the protein is involved in regulation - IP3, ATP, calmodulin binding sites
Very little forms the pore feature of the channel
Binds Ins(1,4,5)P3 (Kd 10nM) - not Ins(2,4,5)P3 or Ins (1,3,4,5)P4 (high specificity)
Ca2+ release is quantal - it is proportional to the [IP3]
Regulated by luminal & cytosolic [Ca2+], ATP, phosphorylation (PKA)
Describe intracellular Ca2+?
Cytosol 0.1mM-1.0mM, whereas blood is 1-2mM
This allows a steep concentration gradient across the membranes
Two reporter groups for Ca2+:
Photoprotein (aequorin)
Fluorescent dyes based on EGTA (Fura-2, Fluo-3) - coordinate the binding of Ca2+, which alters the fluorescence
They are used as calcium sensors to understand calcium signalling
Describe Ca2+ spikes on a graph?
When a cell is loaded with Ca2+ sensitive protein aequorin and exposed to increasing concentrations of vasopressin
There is a [Ca2+] base line and as the concentration increases the frequency of the spikes increase but the amplitude of [Ca2+] stays the same
Describe Ca2+ signal release and influx?
Ca2+ store has finite capacity - as the ER volume is finite and amount of buffer available etc…
IP3 stimulates Ca2+ from the ER - depleting it’s stores
Depletion of Ca2+ prompts entry of Ca2+ through Ca2+ channels into the plasma membrane (ICRAC, Trp), from the blood
Called “capacitative Ca2+ entry”
STIM is on the ER - when the store is empty it moves to the rest of the complex causing the channel TRPC to open
What does calcium interact with?
Ca2+ interacts with many proteins to alter function, sometimes directly (channels, proteases, lipases) other times through regulatory proteins (e.g. calmodulin, annexins)
Describe calmodulin?
Calmodulin (CaM) - ubiquitous, abundant protein
It binds to calcium and interfaces with other target proteins
It is in high concentration = stochiometric interaction
Small 16.8kDa protein and 4 Ca2+ binding sites of EF-hand structure
It affects cyclic nucleotide metabolism, glycogen metabolism, Ca2+ transport and smooth muscle contraction
Describe the interaction of calmodulin and its partners?
Substantial conformational change upon Ca2+ binding (Kd sub-mM)
Extensive hydrophobic domain, rich in met (methionine puddle) and exposed
This is due to the ends coming together
The methionine puddles fold around a target protein to bury the hydrophobicity
Interacts with simple motif in targets containing +ve charged segment (N-terminal) followed by hydrophobic segment (C-terminal) e.g. RRKLKGAITTMLA (CaM KII)
It is looking for patterns but not necessarily a specific target
This changes the behaviour of the target protein
Describe calmodulin control of the cellular event - neurotransmitter release?
Ca2+ increases to release vesicles containing neurotransmitter
Synaptic vesicles in ‘reserve’ are moved down towards the synapse when the previous ones were released
Ca2+ also interacts with actin cytoskeleton and calmodulin - allowing synapsin to be released upon phosphorylation by Calmodulin kinase II
