Second Messengers

Question 1

Give an overview of second messengers - cAMP?

Answer 1

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

Question 2

Describe cyclic AMP?

Answer 2

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)

Question 3

Describe the production of cAMP?

Answer 3

1. Ligand binding alters the conformation of the GPCR, exposing the binding site for Gs protein (alpha, beta and gamma subunits)
2. Diffusion in the bilayer leads to association of ligand-receptor complex with Gs protein = greatly weakening the affinity of Gs for GDP
3. GDP dissociates, allowing GTP to bind - causing the a subunit to dissociate from the Gs complex, exposing the binding site for adenylyl cyclase
4. The a subunit binds to/activates the adenylyl cyclase - now producing many cAMP
5. The dissociation of the ligand then returns the receptor to its original conformation
6. 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)

Question 4

Describe adenylyl cyclase?

Answer 4

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)

Question 5

Describe the cAMP phosphodiesterase?

Answer 5

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

Question 6

Describe the fight/flight response?

Answer 6

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

Question 7

Describe the need of control with cAMP?

Answer 7

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

Question 8

How is cAMP production limited?

Answer 8

Receptor optimised for inactivity - needs a ligand to bind/activate
Receptor internalised via endocytosis into the cell
Ga hydrolyses GTP
GAP activates GTPase activity

Question 9

What are the modes of signalling by cAMP?

Answer 9

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

Question 10

Describe the subunits of cAMP-dependent protein kinase A?

Answer 10

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

Question 11

Describe the structure of cAMP-dependent protein kinase A?

Answer 11

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

Question 12

Give some examples of targets of PKA?

Answer 12

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

Question 13

How is the signal (PKA) encoded/decoded?

Answer 13

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

Question 14

What doe AKAPs do?

Answer 14

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

Question 15

Describe the overuse of signals?

Answer 15

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

Question 16

Give an overview of Ca2+ as a second messenger?

Answer 16

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

Question 17

Describe IP3 and its role?

Answer 17

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)

Question 18

Describe how phospholipase C is activated?

Answer 18

2 major routes - they activate different isoforms of the enzyme
In both - PIP2 is cleaved into IP3 and DAG

1. GPCR (7 helix receptor) with a heterotrimeric G protein
   Ligands can be: acetylcholine, serotonin, 5-hydroxytryptamine or photons of light
2. (platelet derived) Growth factors

Question 19

Describe GPCR signalling to phospholipase C?

Answer 19

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)

Question 20

Describe growth factors signalling to phospholipase C?

Answer 20

Class of receptors that have an enzymatic function on the cytoplasmic surface
Single transmembrane domain with a large extracellular domain for ligand binding

1. The ligand can bind to two receptors - bringing the receptors physically closer together
   Receptors dimerise and autophosphorylate - via kinases and tyrosine kinases
2. SH2 domain of PLCg docks onto phosphoreceptor
3. PLCg phosphorylated (Y771, Y783, Y1254) and activated
   PKA/PKC can phosphorylate and inhibit PLCg

Question 21

Describe the intracellular store of Ca2+?

Answer 21

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

Question 22

Describe the IP3 receptor?

Answer 22

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)

Question 23

Describe intracellular Ca2+?

Answer 23

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

Question 24

Describe Ca2+ spikes on a graph?

Answer 24

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

Question 25

Describe Ca2+ signal release and influx?

Answer 25

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

Question 26

What does calcium interact with?

Answer 26

Ca2+ interacts with many proteins to alter function, sometimes directly (channels, proteases, lipases) other times through regulatory proteins (e.g. calmodulin, annexins)

Question 27

Describe calmodulin?

Answer 27

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

Question 28

Describe the interaction of calmodulin and its partners?

Answer 28

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

Question 29

Describe calmodulin control of the cellular event - neurotransmitter release?

Answer 29

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