BS3054 L6 - Cyclic Nucleotides

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)

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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