Cell Signalling Flashcards
Describe how cell activity is affected.
- Most things which affect cell activity or function do not enter the cells.
- They act on membrane-bound receptors that control signalling proteins via the production of second messengers.
- These mediate cell activity.
What are the general principles of signal trasduction?
- Many signalling proteins act as molecular switches.
- There are two common ways to activate / deactivate signalling proteins.
- Human genomes encodes ~520 kinases and ~150 phosphatases.
- There are two main types of kinases.
- There are two types of GTP-binding proteins.
What are the 2 main ways of switching signalling proteins on/off?
- Phosphorylation
- GDP binding
What are the 2 main types of kinases?
- Tyrosine kinase
- Serine / threonine kinase
What are the 2 types of GTP-binding proteins?
- Trimeric G proteins
- Monomeric GTPases
Explain the role of G-protein-coupled receptors in signal transduction.
- 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 is a G-protein-coupled receptor?
- All G-protein-coupled receptors have 7 membrane spanning regions with their amino termini on the extracellular face and their carboxy termini on the cytoplasmic face of the plasma membrane.
- The 7 membrane-spanning regions are alpha helices.
- A 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.
- G-protein activation and complex formation are part of a cycle.
- Trimeric: composed of 3 different subunits (α, β and Ɣ)
What is the mechanism of action of a G-protein-coupled receptor?
- Binding of the ligand to the receptor changes its conformation, causing it to bind to the Gα protein in such a way that GDP is displaces and GTP is bound.
- This triggers Gβɣ dissociation activating downstream pathways.
- Activation is short-lived, as GTP bound to Gα hydrolyses to GDP in seconds, leading to the re-association of Gα with Gβɣ and inactivation of adenylate cyclase.
Describe how adenylate cyclase is stimulated.
- GTP is required for the ligand-induced stimulation of adenylate cyclase.
- Glucagon receptor couples to GαS.
- Overall, the system needs:
- A receptor
- A transducer (G-protein)
- An amplifier (adenylate cyclase) that generates large amounts of a second messenger.
- cAMP is the second messenger made by adenylate cyclase.
Describe the structure of a G-protein-coupled receptor.
- Receptors consist of 7 transmembrane helices (hydrophobic amino acids) that reside in the plasma membrane.
- Interacts with heterotrimeric G-protein complex on the extracellular side.
Which type of G-protein stimulates phospholipase C?
Gαq
Which type of G-protein stimulates adenylate cyclase and increases cAMP?
Gs
Which type of G-protein inhibits adenylate cyclase and decreases cAMP?
Gi
What are the functions of the Gβ/ɣ dimer?
- Gate ion channels
- Stimulates adenylate cyclase
- Stimulates PLA2
- Stimulates PLC-β, PLC-ε and PLC-𝜼
How does phospholipase C become activated?
Its substrate is phospholipis which resides in the cell membrane.
1-2% of the cell membrane is this phospholipid so it is not abundant.
What are phospholipase C isoforms?
- Proteins which possess distinct domain structures but catalyse the same reaction (liberation of IP3 and DAG from PIP2).
- Some domains are common (catalytic, membrane localisation).
- Some domains are uique (regulatory).
- Acivated by different pathways.
Describe calcium signalling.
- Cytosolic calcium levels are dynamic.
- Calcium can enter from intracellular stores or from outside the cell via calcium channels.
- Channels may be receptor or voltage operated.
- Calcium can exit via other channels present in plasma or organelle membranes.
What is the resting calcium concentration of a cell?
~100nM
What is the activated calcium concentration of a cell?
0.5-1µM.
Cellular response depends upon the duration of the signal.
What is the effect of the calcium wave on an egg cell?
- Calcium wave does 2 things:
- Tells the cell to start dividing
- Prevents any more sperm coming in because an egg should not be fertilised
Describe protein kinase C.
- A large family: at least 12 different isoforms.
- Most are present as catalytically inactive, soluble proteins in the cytoplasm.
What happens to PKC in the cell?
- 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
Protein kinase C functions ‘indirectly’ to do what?
To alter gene expression.
The genes can phosphorylate kinases.
Binding of adrenalin to the β2 adrenergic receptor is another type ot G-protein-coupled receptor signalling.
Describe what happens.
- 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
- Bindind of adrenaline to β2 adrenergic receptor increases the intracellular concentration of cAMP (cyclic AMP) as receptor couples to Gαs.
- cAMP is synthesised within cells from ATP by the enzyme adenylate cyclase.
- cAMP is degraded by the enzyme cAMP phosphodiesterase.
- Different receptors utilise a common adenylate cyclase (ie each receptor does not have its own intrinsic adenylate cyclase).
How does increased cAMP activate the cAMP-dependent protein kinase, PKA which is downstream?
Describe the effect of signal transduction.
- Signal transduction results in an amplification of a signal:
- A small change at the top can have a large change at the bottom.
- A large amount of product is made relative to the amount of reactants. A small amount of signal can generate a large amount of second messenger within the cell.
What can the catalytic subunits of PKA do?
The catalytic subunits of PKA can phosphorylate sibstrates on serine or threonine residues. It has substrates in the membrane, cytoplasm and the nucleus.
What does PKA do in the nucleus?
- In the nucleus, PKA can activate transcription of genes containing cAMP response elements, or CREs in their promoter.
- A specific transcription factor, the cAMP response element binding protein, CREB binds to this sequence and activates transcription of downstream genes.
- When CREB is unphosphorylated, it is inactive; only in its phosphorylated state does CREB activate transcription.
What is the function of CREB?
- A specific transcription factor, the cAMP response element binding protein, CREB binds to this sequence and activates transcription of downstream genes.
- When CREB is unphosphorylated, it is inactive; only in its phosphorylated state does CREB activate transcription.
Describe mechanism of action of the cholera toxin.
- Oligomeric complex which after cleavage, becomes active and enters intestinal epithelial cells to stimulate Gαs.
- The overstimulation of cAMP production results in a release of water and ions including Na+, K+, Cl- and HCO3- into the intestine.
- This leads to rapid fluid loss and dehydration.
What are receptor tyrosine kinases (RTKs)?
- High affinity cell surface receptors for many polypeptide growth factors, cytokines and hormones.
- Enzyme-linked receptor.
- Example: insulin-like growth factors activate RTKs to control cell proliferation.
- RTKs are associated with cancer because they control cell proliferation - in cancer there is often uncontrolled cell proliferation.
What is Ras?
- Ras superfamily contains over 100 members: Rho, Rap, Rab, Arf etc.
- They regulate cellular processes: proliferation, cytoskeletal dynamics, membrane trafficking / vesicular transport.
What is the role of GTPases in disease?
- Damage to small GTPase switches can have catastrophic consequences for the cell and the organism.
- Several small GTPases of the Rac/Rho subfamily are direct targets for clostridial cytotoxins.
- Further, Ras proteins are mutated to a constitutively-active (GTP-bound) form in approximately 20% of human cancers.
- They bind GTP and cannot release / hydrolyse it again. This is associated with cancerous cells.
What does Ras activate?
- The mitogen-activated protein kinase (MAPK) pathway.
- Ras activates Raf (kinase), which in turn stimulates gene transcription via other kinases (MEK and ERK).
What is EGFR?
EGFR (epidermal growth factor receptor) is an RTK (receptor tyrosine kinase) activated by TGFα (transforming growth factor alpha).
What does the EGFR pathway do?
The EGFR activates Ras via Grb2/SOS proteins.
What is associated with mutations in EGFR, Ras and Raf?
Tumorigenesis because they cause overexpression and/or hyperactivation of the respective proteins.
FOCUS ON THESE PATHWAYS FOR THE EXAM
How does the pertussis toxin work?
- Pertussis toxin works in the reverse manner to the cholera toxin.
- It Inhibits Gαi in order to increase cAMP production in lung epithelia.
- Pertussis toxin - whooping cough.
