BS3054: TOPIC THREE

Secondary Messengers

Intermediate chemicals that help transduce a chemical signal

- e.g. cAMP, cGMP, inositol triphosphate, Ca2+, diacylglycerol

What is cAMP

- second messenger

- product of reaction catalysed by adenylyl cyclase

- ATP (+ adenylyl cylase) --> cAMP

Adenylyl cyclases

- 10 isoforms

- M1 and M2 membrane bound domains

- C1 and C2 domains in the cytosol

- ATP binding site sits in-between C1 and C2 domain

- Mg2+ binding site = Mg2+ is a cofactor

- activity dependent on the Galpha subunit it receives signals from

How do isoforms of adenylyl cyclase cause specifictiy

- different isoforms respond differently to signals from G-proteins

- allows tissue specificity - not all tissues will respond to a signal and some will respond stronger than others

effectors of cAMP mediated signalling

- Protein Kinase A

- cyclic nucleotide gated channels

- cyclic nucleotide regulated GEFs

What is PKA

- protein kinase A

- serine-threonine kinase

- phosphorylates proteins

- modulated by cAMP

Structure of PKA

- two regulatory subunits

- two catalytic subunits

- 4 isoforms of R subunit (RI-alpha, RI-beta, RII-alpha, RII-beta)

- 3 isoforms of C subunit (C-alpha,beta,gamma)

w

What happens when cAMP binds PKA

- cAMP binds to regulatory subunit dimer = releases the catalytic subunits from the complex

- the catalytic subunits are now active and can phosphorylate

What are the two classes of PKA

PKAI

PKAII

PKA I vs PKA II

PKA I - cytosolic

- cAMP binds to PKA and causes dissociation of the catalytic subunit = can phosphorylate substrate

PKA II - membrane bound

- docked to AKAP

- substrate binds to AKAP and then cAMP causes release of the R subunits = allows substrate to be phosphorylated

- AKAP localises PKA to specific cellular targets

What is AKAP

- A-kinase anchoring protein

Examples of PKA targets

- GPCRs, ion channels, cytoskeletal proteins, protein phosphatase and kinase inhibitors, transcription factors

Phosphodiesterase

enzyme that degrades cAMP, producing 5'AMP, to terminate signalling

- breaks phosphodiester bond

- regulated by kinases

Are there only one type of PDE (phosphodiesterase)

- 11 different families of PDEs in mammals

- some hydrolyse both cGMP and cAMP

- some preferentially hydrloyse cAMP or cGMP

guanylyl cyclases

- guanylyl cyclase catalyses GTP to cGMP

- PDE will convert cGMP to 5'GMP

What are the two types of guanylyl cyclase

1. particulate GCs = membrane bound

2. soluble GCs = in cytosol/ NO sensitive

particulate guanylyl cyclases

- transmembrane, ligand-activated homodimers

soluble guanylyl cyclases

- activated by NO and CO

- NO is an extremely potent activator

- nitric oxide binds haem group in guanylyl cyclase heterodimer

Nitric oxide

- vasodilator

- gaseous compound - only stable for seconds so made as and when needed

- detectable at very small amounts

NOS

- nitric oxide synthase

- synthesises NO

types of NOS

nNOS = neuronal + skeletal muscle = communication

iNOS = inducible = produces high NO concentrations that can exhibit direct toxic effects = immune defence

eNOS = endothelial = vasodilation

biosynthesis of Nitric oxide by NOS

- L-arginine is turned into L- citrulline and nitric oxide by NOS

Roles of NO

- activates soluble guanylyl cyclases

- nitrosylation of proteins

- direct toxicity (NO is a free radical)

major targets of cGMP

- cyclic nucleotide gated channels

- modulation of PDE activity

- activation of PKG

what is PKG

cGMP dependent protein kinase

PKG monomer

Regulatory domain

- Leucine zipper (pseudo-substrate)

- Nucleotide binding sites

Catalytic domain

- ATP site

- Kinase

= exist as a soluble homodimer

what is a pseudo-substrate

- substance that mimics the real substrate of an enzyme

- often part of enzyme own structure

- blocks enzyme active site and inhibits activity

How does NO cause vasodilation

- NO activates soluble guanylyl cyclase

- increases levels of cGMP within smooth muscle cells of blood vessel walls

- rise in cGMP leads to activation of PKG

- PKG activates SERCA pump to move calcium ions from cytoplasms to ER

- cGMP can activate potassium channels causing hyperpolarisation = closes VGCC = promoting relaxation

Phospholipid structure

2 fatty acids, 1 glycerol, 1 phosphate group

- hydrophilic head and hydrophobic tail = amphipathic

X group on phosphate of phospholipids

- can add groups

e.g. add choline to make phosphatidylcholine

e.g. add inositol to make phosphatidylinositol

phospholipids classified according to their polar head group and their abundance

- phosphatidylcholine = 50% of membrane lipids

- phosphatidylserine = 2-10%

- phosphatidylethanolamine = 15-35%

- phosphatidylinositol = 5-10%

Other denotations of phosphatidylinositol

PI or PtdIns

How is PI modified

- phosphorylation of any of the 6 carbons on the inositol group make other signalling molecules e.g. PIP2

Variations of PI

- phosphatidylinositol = PI

- phosphatidylinositol 4-phosphate = PIP

- phosphatidylinositol (4,5) - bisphosphate = PIP2

- phosphatidylinositol (3,4,5) - triphosphate = PIP3

why is PIP2 called phosphatidylinositol 4,5-bisphosphate not called phosphatidylinositol 4,5-diphosphate

- because the phosphates are on different carbons they are not organised in a chain where it would be referred to as diphosphate

Which carbons is PIP2 phosphorylated on

carbons 4 and 5

phosphatidylinositol structure

what enzymes turn phospholipids into signalling second messenger molecules

phospholipases

variations of phospholipases

- PLA1

- PLA2

- PLD

- PLC

= same substrate different outcomes

Role of phospholipase C

- cleaves PIP2 between the oxygen on the glycerol backbone and the phosphate

- forms DAG and IP3 = both second messengers

what is IP3

inositol 1,4,5-triphosphate

Receptor mediated signalling through PLC

- agonist binds receptor = conformational change

- alpha subinit dissociation and GDP/GTP exchange

- recruitment of PLC = cleaves PIP2 = DAG + IP3

- IP3 stimulates release of calcium from IC stores (ER) by binding to IP3 receptor

- DAG and calcium activates PKC

IP3 receptor

- acts as coincidence detector

- requires both IP3 to bind and calcium to be present for ion channel to open and cause calcium release into cytoplasm = calcium induced calcium release

families and isoforms of PLC

6 families, 13 isoforms (30 splice variants):

- PLC-beta (4 isoforms)

- PLC - gamma (2 isoforms)

- PLC - delta (3 isoforms)

- PLC - epsilon (1 isoform)

- PLC - zeta (1 isoform)

- pLC - eta (2 isoforms)

why is it important for there to be different isoforms of PLC

- to allow precise control so signalling can be regualted between cell types/locations

PLC-beta

- has 4 isoforms PLC-b1-4

- X and Y catalytic domains

- PH domain = allows localisation = bind PIs

- 4 tandem EF-hand domains = calcium function but unclear

- C2 domain = Ca2+ binding

- CC domain and PDZ domain = protein-protein interactions with PLC

homology between isoforms of PLC-beta

- 60% homology of the catalytic domain between isoforms

what is PLC-b most commonly associated with regulating

GPCRs

PLC-beta isoforms tissue distribution

B1 and B3 = fairly widespread

B2 = immune/haematopoietic

B4 = retina and certain neurons

What can activate PLC-beta

- Gaq subunit

- G beta-gamma subunit

- Ca2+

what domains does G-beta-gamma subunit associate with on PLC-beta

- PH domain (localisation of PIs)

- catalytic region

- almost always beta-gamma subunit from Gai/10 proteins as they are the most abundant

PLC-beta role other than phospholipase activity

- GTPase activating proteins

- can bind to alpha subunit and speed up intrinsic GTPase activity to cause reassociation between alpha and beta-gamma subunit

- same role as RGS proteins

coincidence detection with G-proteins and PLC-B3

- Gaq and Gbeta-gamma both activate PLC-B3 = synergistic activation

how is inositol recycled

- dephosphorylation of IP3 creates inositol

- inositiol fed back into membrane where it is phosphorylated into PIP2 again by kinases

- PIP2 can be turned into IP3 and DAG again or phosphorylated by phosphoinositide 3-kinase into PIP3 = signalling

phosphoinositide kianses

- phosphorylate PIs at 3,4,5 positions on inositol

- phosphatases dephosphorylate these

e.g. phosphoinositide 3-kinase phosphorylates in the 3 carbon position

PI3Ks

= phosphatidylinositol 3-kinases

= phosphorylate in the 3-OH position of the inositol ring in PIs

- 3 main classes = I, II, III

- activated by diverse cell surface receptors mainly RTKs and some GPCRs through Src transactivation of RTKs

- its preferred substrate in vivo is PIP2 = converts it to PIP3

GPCR singalling via Src transactivation of RTKs

- agonist binding to GPCRs can cause Src activation

- Src can then phosphorylate and activate RTKs

- transactivation amplifies the signal by integrating different pathways onto the RTK cascade

Structure and function of PI3-kinase

Regulatory subunit:

- p85 with SH2 and SH3 domains associated = allows recruitment to RTKs or adaptor proteins

Catalytic subunit

- p85 binding domain

- Ras binding domain - Ras binding can activate catalytic subunit

- HR3 = membrane binding

- HR2 = scaffold for other proteins to bind to it

- HR1 = kinase core

Why is PI3k associated with cancer

- Ras binding to PI3k causes cell proliferation

activation of PI3 kinase via RTKs

- growth factor binds to RTK causinf autophosphorylation and dimerisation

- allows docking of Grb2, SOS, RAS and GTP

- PI13k docks via Ras domain

= production of PIP3 on membrane

PIP3 as an anchor for signalling proteins

- signalling proteins with PH domains accumulate at sites of PI3K activation by binding to PIP3

- these proteins regulate cell growth, survival and movement

- examples of proteins containing PH domains are PKB (Akt) and PDK1

PKB (Akt)

- serine/threonine kinase

- growth factor pathway

- activated by PI3K and PDK1/2

PDK1

phosphoinositide dependent kinase 1 (involved in activation of PKB)

PKB activation via PDK1

- PIP3 in the membrane recruits PKB (Akt) and PDK1

- PDK1 phosphorylates PKB to partially activate it

- mTORC2 complex further phosphorylates AKT to fully activate it

- activated AKT then inhibits the TSC complex leading to activation of mTORC1 = controls protein synthesis and growth

What is PKB/Akt generally associated with

anti-apoptosis, growth, proliferation and migration

Termination of PI3-kinase signalling

- SHIP proteins remove binding sites for proteins with PIP selective PH domain

- SHIP proteins generate PIP2 from PIP3 that PKB can bind to

- PH domain of PKB binds PIP2 and PIP3 with equal affinity

- PTEN turns PIP3 into PIP2 and PIP2 into PI

PI3-kinase signalling and cancer

- numerous oncogenes activate type IA PI3-kinase

- activating mutation of PI3-kinase described in cancer

- PTEN has tumour supressor properties - mutations in PTEN associated with cancer

- mutations in SHIP1 recently associated with some leukaemias

therapeutic attempts to inhibit PI3-kinase in cancers

- small non-specific molecules

- wortmannin

- LY294002

- copanlisib and apelisib - only active on one kinase (class I PI1K inhibitors)

- most approved drugs have since been withdrawn due to side effects

Theoretical ideal therapeutic targets for PI3K in cancer

Isotype selective PI3K inhibitors:

- inhibitors that target specific p110 catalytic subunits - many minimise side effects

Inhibitors of Akt (not yet apporoved)

- inhibition of downstream signalling from PI3K activation many be beneficial

- two examples of Akt inhibitors:

1. ipatasertib = binds ATP binding site of Akt (breast cancer)

2. afuresertib = competitive inhibitor

Why is calcium important to the body?

- strong bones and teeth

- fertilisation

- proliferation

- neurotransmission

- apoptosis

- contraction

- gene expression

Intracellular vs extracellular calcium conc

- Calcium concentration is much higher outside the cell than in the cell cytoplasm

- 1-2mM (10^-3) outside

- 100nM (10^-7) inside

What is the equation for the driving force of calcium movment

Nernst equation

E = RT/zF ln ([ion outside] / [ion inside])

Advantages of IC calcium conc being much lower than EC

- this is good because it means only a small amount of calcium needs to move into the cell in order to create a large change

- the balance can also be restored easily if not much calcium has to move

Disadvantages of IC calcium conc being much lower than EC

- to remove ions from the cell requires a lot of ATP

- cell can become overloaded with calcium easily which leads to cell death

How is the calcium gradient set up and maintained

1. impermeability of membrane

2. Ca2+ release across plasma membrane

- Ca2+-ATPase

- Na/Ca exchanger

3. Ca2+ buffers

4. Intracellular Ca2+ stores

1. Impermeability of membrane in calcium gradient set up

- hydrophobic lipids in the membrane = relatively impermeable

- once calcium has left cell it is hard for it to get back in

= maintains higher conc outside than in cell

2. Ca2+ release across plasma membrane in calcium gradient set up

Ca2+ ATPase (PMCA) - plasma membrane calcium atpase

- sits in plasma membrane

- drives calcium out of cell using ATP

- high affinity so can bind even with low levels of calcium inside cell

- low capacity = saturates quickly so not very efficient

Na+/Ca exchanger

- electrochemical gradient of Na+ means that Na+ moves from outside of cells to inside

- 3 Na+ move into the cell and take 1 Ca2+ with them

= antiport system

- this system is electrogeneic = works best at resting membrane potential

- low affinity but high capacity

Ca2+-ATPase pump (PMCA)

- calcium conc increases in the cell

- calcium binds to calmodulin and activates it

- calmodulin affinity for calcium increases upon calcium binding

- caclium-calmodulin binds to Ca2+-ATPase

- Ca2+-ATPase removes calcium from the cytoplasm into the EC space

3. Calcium buffers in calcium gradient set up

- when calcium enters the cell it doesn't travel far

- it gets tied up in calcium binding proteins e.g. parvalbumin, calreticulin

- calcium will only travel through if these proteins are saturated when there is a large calcium influx

- regulates the amount of calcium needed to initiate a cellular response

4. Intracellular stores of calcium in gradient set up

- calcium is stored in the SR/ER of the cell

- IP3 binding to receptors or Ca2+ binding to ryanodine receptors (calcium induced calcium release) = causes calcium release from ER into cytoplasm

- SERCA pump reuptakes calcium into SR/ER

How are calcium levels elevated and returned to basal levels

1. influx across plasma membrane

- voltage-gated Ca2+ channels

- inotropic receptors = ligand-gated calcium channels

2. rapid release from EC/SR

3. Golgi, nucleus and mitochondria (non-rapid) can also serve as Ca2+ stores

How is ca2+ influx across the membrane regulated

VOCCs

- channels sense membrane depolarisation and change shape = opens

- negative amino acid residues on the channel attract positive calcium = calcium moved through the channel into the cell along conc gradient

Ligand-gated ion channels (inotropic)

- ligand binds receptor and causes channel to open

- calcium will flow into the cell along concentration gradient

e.g. ACh binding receptors allowing Ca2+ influx

How is Ca2+ release from IC stores regulated

Release into cytoplasm from ER:

- GPCR activation

- Activation of PLC

- PIP2 -> DAG and IP3

- IP3 binds receptors on ER and stimulates Ca2+ release into cytoplasm

= ligand-gated ion channel but on ER membrane not plasma membrane

- Ca2+ now in the cytoplasm opens ryanodine receptors on ER membrane which causes further release of calcium from the ER = calcium induced calcium release

Re-uptake into the ER = SERCA pump

- Ca2+-ATPase that pumps Ca2+ from the cytoplasm into the ER/SR

IP3 and ryanodine receptors on SR/ER

- regulated by calcium

- when calcium is low it stimulates them to open

- at high calcium conc in cytoplasm ryanodine and IP3 receptors are inhibited

Why are there calcium binding proteins inside ER

- it calcium reacts with phosphates it forms a solid

- binding proteins allow high levels of Ca in ER without it solididying

IP3 receptors

- tetramer

- 4 subunits

- ligand gated ca2+ channel

- IP3 binds outside

Why is calcium stored in nucleus

- the nuclear envelope is continuous with the ER

Non-rapid releasable store of calcium - mitochondria

- when calcium levels are very high mitochondria will take up calcium to protect the cell from death

- microdomains = if calcium flood into the cell close to the mitochondria then that site will be highly concentrated = localised high concentration of calcium

= the mitochondria will take up the calcium

- mitochondria dont release calcium easily

What is mitochondrial calcium uptake driven by

- large negative inner membrane potential

- voltage dependent anion channels

- ca2+ uniporters of inner membrane

Why do mitochondria take up calcium

- calcium buffering

= regulate calcium levels if in excess - can release later and more slowly

- stimulation of mitochondrial metaboism

= calcium overload in cells requires mito to produce more ATP to power pumps to remove calcium

- cell death

= avoids cell death from overload of calcium

small calcium stores

lysosomes, endosomes, phagosomes, secretory vesicles

Store refilling and restoration of calcium concentration

- too much calcium for too long is toxic so levels have to return to basal

= termination of signal (desensitisation or ligand dissociation/reuptake)

= calcium removal

= Ca2+ store refilling

How are calcium stores refilled

- recycling of cytosolic calcium that was released from ER

- capacitative Ca2+ entry

Capacitative Ca2+ entry

- capacitative or store-operated channel (SOC)

- system that tells the cell that the ER stores are depleted

- SOC sits in the membrane and interacts with the SERCA pump

- STIM1 sits in ER membrane = detects ER stores are low and binds calcium = activation of STIM1

- STIM1 aggregates together and localises by the CRAC channels = CC domains on CRAC channels and STIM1 interact

- = CRAC channels open = allow calcium back into the ER

Synaptotagmin

- calcium sensor that affects NT vesicle release

- synaptotagmin sits in membrane of vesicle and binds calcium - when bound to calcium it has higher affinity for syntaxin

- C2A and C2B domains interact with syntaxin on the plasma membrane

- allows fusion of vesicles to the membrane for NT release

How does calcium regulate so many different processes

SPACE

- dependent on location of influx - from stores or plasma membrane

TIME

- long exposure to calcium avoided by low amplitude signals to make calcium release slow and transient

- depending on the receptors and the tissue types, calcium release and uptake is different

AMPLITUDE

- strength of signal impacts response