Signal Transduction Flashcards
State the function of second messengers
Mediate cell activity - inside the cell
What do signalling proteins act as ?
Molecular switches
State the 2 ways to activate/deactivate signalling proteins
Signalling by phosphorylation
Signalling by GTP binding
What does the human genome encode ?
520 kinases
150 phosphatases
Function of kinase
Unphosphorylated protein - OFF protein
Kinase uses ATP to phosphorylate protein which will change the chemistry of the protein and put it in an ON position.
How does the signalling protein return to its OFF position ?
Cleavage of the phosphate group by another type of enzyme called a phosphatase.
Commonly used mechanism for switching on and off proteins.
Describe the use of GDP and GTP in cell signalling
In OFF position, the proteins will have GDP bound.
Some sort of change in behaviour occurs, such that GDP no longer binds, and its affinity for GTP increases.
GTP binds instead, giving rise to a different structure putting it in the ON position.
State the 2 types of GTP-binding proteins
Trimetric G proteins
Monometric GTPases
Trimeric G proteins
Associated with G protein coupled receptors
State the 2 main types of kinase
Tyrosine kinase
Serine/Threonine Kinase
Describe G-protein coupled receptors
These receptors are coupled to the G proteins, which are hetrotrimeric
These G proteins can open ion channels / activate enzymes which produce second messenger molecules.
Second messenger molecules mediate cellular effects.
Describe how G-protein coupled receptors work
Ligand binding activates a G-protein which in turn activates or inhibits another protein.
Often this is an enzyme that generates a specific second messenger.
What happens when a ligand binds to the G protein coupled receptor ?
Ligand binding to a G protein coupled receptor activates the associated G protein which in turn inhibits / activates a downstream enzyme to generate an intracellular second message.
Describe the structure of G protein coupled receptors
7 membrane spanning regions (alpha helixes) with their amino termini on the extracellular face and their carboy termini on the cytoplasmic face of the plasma membrane.
Describe the intracellular G protein coupled receptor loops
Many cell-surface receptors are coupled to trimeric signal-transducing G proteins that bind either GTP or GDP.
Trimeric
Composed of 3 different subunits (alpha, beta and gamma)
Describe the mechanism of action of G proteins
Binding of the ligand to the receptor changes its conformation, causing it to bind the G alpha protein in such a way that GDP is displaced and GTP is bound.
This triggers G beta/gamma dissociation activating downstream pathways.
Activation is short-lived, as GTP bound to G alpha hydrolyses to GDP in seconds, leading to the reassociation of G alpha with G beta/gamma and inactivation of adenylate cyclase.
Function of GTP
GTP is required for the ligand-induced stimulation of adenylate cyclase.
Glucagon receptor couples to G alpha
What does the Martin Rodbell experiment require ?
A receptor (glucagon)
A transducer (G-protein)
An amplifier (adenylate cyclase - generates large amounts of a second messenger)
Second messenger is cAMP
Describe the structure of GPCR (G-protein coupled receptor)
7 TM helices that reside in the plasma membrane.
Interacts with hetrotrimeric G protein complex on the intracellular side. (alpha, beta and gamma subunits)
G alpha q
Stimulates phospholipase C
G s
Stimulates adenylate cyclase
Increases cAMP
G i
Inhibits adenylate cyclase (basal activity)
Decreases cAMP
Describe G-protein
Subunits
Alpha subunit - bind GTP or GDP
(20 types of G alpha subunit)
Beta gamma dimer - Interacts with beta subunits to regulate G alpha activation
(6 G beta’s and 12G gamma’s)- 72 combinations.
Function of the G beta/gamma dimer
What does it stimulate ?
Gate ion channels
Stimulates PLA2
Stimulates adenylate cyclase
Stimulates PLC-beta, PLC-beta etc.
Resting calcium concentration (in cytosol)
100 nM
Activated calcium concentration (in cytosol)
0.5-1 microM
What does cellular response depend on ?
Duration of signal
- Exocytosis
- Contraction
- Metabolism
- Gene transcription
- Fertilisation proliferation hypertrophy
IP3
Receptor operated channel
Describe calcium in fertilisation
Calcium waves triggers :
- Start of embryonic development
- Prevents other sperm from entering the cell
What is PKC ?
Not just 1 protein, rather a family of proteins (at least 12 different isoforms).
State some features of the 12 PKC isoforms
Most are present as CATALYTICALLY INACTIVE, soluble proteins in the cytoplasm.
Describe the action of PKC
Rise in cytosolic calcium levels causes PKC to bind to the cytosolic leaflet of the plasma membrane, where it can be activated by the membrane-associated DAG and/or Ca2+.
PKC then phosphorylates a wide variety of substrate proteins on serine and threonine residues.
What does activation of PKC function indirectly to do ?
Alter gene expression
Function of binding of adrenalin to the Beta 2 adrenergic receptor
Mediates the body’s response to stress/fear (fight or flight)
- release of glucose and fatty acids from liver/fat cells
- increased contraction of cardiac muscle
Describe binding of adrenalin to the Beta 2 adrenergic receptor
Binding increases the intracellular concentration of cAMP as receptor couples G alpha s.
cAMP is synthesised within cells from ATP b the enzyme adenylate cyclase.
cAMP is degraded by the enzyme cAMP phosphodiesterase.
Describe the structure of the cAMP dependent protein kinase
2 regulatory subunits
2 catalytic subunits
What does signal transduction result in ?
Amplification of a signal.
A small change on top can have a large change at the bottom.
Where are PKA’s substrates found ?
The membrane, cytoplasm and nucleus
How does cholera toxin work ?
Toxin which works by hijacking the g-protein coupled receptor signalling machinery.
Describe the structure of the cholera toxin
Oligomeric complex, which after cleavage becomes active and enters intestinal epithelial cells to stimulate G alpha s.
Describe the action of cholera toxin
Overstimulation of cAMP production results in a release of water and ions into the lumen of the small intestine.
This leads to rapid fluid loss & dehydration.
State the toxin which acts in a reverse manner to cholera toxin
Pertussis toxin inhibits G alpha I
Increases cAMP production
Describe the action of kinase linked receptors
Receptors exist as pairs, signalling molecules tend to be dimers as well.
Dimer bind to a pair of receptors, bringing them closer together.
They target each other, resulting in a self-phosphorylation of the complex.
Phosphorylated sites are then recognised by intracellular signalling molecules within the cell.
Small GTPases
Single proteins
e.g. RAS
Regulates cellular processes : proliferation etc.
What happens as a result of damage to GTPases ?
Catastrophic consequences for the cell and organism.
What are several small GTPases of Rac/Rho subfamily direct targets for ?
Clostridial cytotoxins
Ras proteins
Mutated to a constitutively active form in approximately 20% of human cancers.
What pathway is associated with cancer ?
EGFR receptor pathway
