Signal Transduction Flashcards
What do many signalling proteins also act as?
molecular switches
What are the two common ways to activate/deactivate signalling proteins?
By phosphorylation
By GTP binding
How many kinases/phosphatases does the human genome code for?
~520
kinases and
~150 phosphatases
What are the two main kinds of kinases?
- tyrosine kinase
- serine/threonine kinase
What are the two types of GTP-binding proteins?
- Trimeric G proteins
- Monomeric GTPases
What process occurs at G-protein coupled receptors (GPCRs)?
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.
How is an intracellular second message generated?
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.
Describe the structure of 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.
What can trimeric signal transducing G proteins bind?
Many cell-surface receptors are coupled to trimeric signal-transducing G proteins that bind either GTP or GDP
What does trimeric mean?
composed of three different subunits (a, b and gamma in G proteins - beta and gamma split off from alpha to activate it)
What is the mechanism of action of a trimeric G protein in a G protein coupled receptor?
Binding of the ligand to the receptor changes its conformation, causing it to bind to the G(alpha) protein in such a way that GDP is displaced and GTP is bound.
This triggers G(b+g) dissociation activating downstream pathways.
Activation is short-lived, as GTP bound to G(alpha) hydrolyzes to GDP in seconds, leading to the re-association of Ga with Gbg and inactivation of adenylate cyclase.
What is GTP required for as discovered by Martin Rodbell?
GTP is required for the ligand-induced stimulation of adenylate cyclase.
What does the glucagon receptor bind to?
Glucagon receptor couples to Gα(small)s
Overall, what does a signal transduction system need?
A receptor
A transducer (G-protein) and
An amplifier (adenylate cyclase) that generates large amounts of a second messenger.
What is GPCR structure?
Receptors consist of 7 TM helices (hydrophobic amino acids) that reside in the plasma membrane
Interacts with heterotrimeric G protein complex on the extracellular side
What part of GPCR crosses the plasma membrane?
The receptor, eg;
β2 adrenergic receptor
7TM receptor
What part of GPCR is the G protein signalling complex?
α, β and γ subunits
How many types of G proteins are there?
20
What are all the types of G proteins?
(All not greek script is subscript) Gαq (or Gq) Gs Gi G12/13 Golf Gt Gβ/γ dimer
What are the important three G protein types and what do they do?
Gαq (or Gq) → stimulates phospholipase C
Gs → stimulates adenylate cyclase, increases cAMP
Gi → inhibits adenylate cyclase, decreases cAMP
What does Gαq (or Gq) do?
stimulates phospholipase C
What does Gs do?
stimulates adenylate cyclase, increases cAMP
What does Gi do?
inhibits adenylate cyclase, decreases cAMP
What does G12/13 do?
regulates cytoskeleton, cell junctions, movement, stimulates PLC-ε
What does Golf do?
activates olfactory receptors
What does Gt do?
transduces visual signals with rhodopsin in the retina
What does a Gβ/γ dimer do?
→ gate ion channels
→ stimulates PLA2
→ stimulates adenylate cyclase
→ stimulates PLC-β, PLC-ε and PLC-η
How many Gβ’s and Gγ’s are there?
6 Gβ’s and 12 Gγ’s
72 combinations
What do all GPCRs exhibit?
preferential association with a particular G protein
or subset of G proteins
What does GPCR stand for?
G protein-coupled receptor
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 unique (regulatory)
How are phospholipase C isoforms activated
Different pathways including GPCR activation (Gαq) Receptor tyrosine kinases Kinases (possibly ↑Ca2+) Small GTPases Fertilisation ↑Ca2+, Gβγ, other mechanisms?
What is the normal calcium level found in the cytosol?
Trick question bitchboy: cytosolic calcium levels are dynamic
How does calcium enter/exit the cytosol?
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 in the cytosol?
~100 nM
What is the activated calcium concentration in the cytosol?
Activated calcium concentration = 0.5-1 μM
Why is the activated cytosolic calcium concentration variable?
Cellular response depends upon duration of the signal
What biological processes can calcium signals trigger
Many,
an example:
fertilisation of an egg by a sperm – initial spark by PLC-ζ (zeta) triggers opening of surface calcium channels
Calcium wave triggers start of embryonic development and prevents other sperm from entering the cell
How many different isoforms does protein kinase C have?
At least 12 probably
How are most protein kinase C-s (PKCs) found in the cell
Most are present as catalytically inactive, soluble proteins in the cytoplasm
What causes the activation of protein kinase C?
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+.
What does activated protein kinase C do?
PKC then phosphorylates a wide variety of substrate proteins on serine and threonine residues.
What other functions does protein kinase C have?
PKC has substrates in the cytoplasm and some isoforms can translocate to the nucleus to phosphorylate nuclear proteins, and can thus function in a transient way or in a more permanent way (gene transcription).
Protein kinase C activation can also function “indirectly” to alter gene expression
Provide an example of other types of GPCR signalling
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
Binding of adrenalin to b2 adrenergic receptor increases the intracellular concentration of cAMP (cyclic AMP) as receptor couples to Gs
cAMP is synthesized within cells from ATP by the enzyme adenylate cyclase
cAMP is degraded by the enzyme cAMP phosphodiesterase
Different receptors utilize a common adenylate cyclase (i.e., each receptor does not have its own intrinsic adenylate cyclase).
How does increased cAMP activate the cAMP-dependent protein kinase, PKA?
cAMP binds to regulatory subunits bound to catalytic subunits, causing a conformational change which releases the catalytic subunits, thus exposing the catalytic site
What does signal transduction do to a signal?
It amplifies it
What can the catalytic subunit of PKA phosphorylate?
substrates on serine or threonine residues
Where does PKA have substrates?
in the membrane, cytoplasm and the nucleus.
What does PKA do 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.
i.e. ligand binding to a cell surface receptor can induce gene expression
What is the cholera toxin and what does it do?
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 lumen of the small intestine
This leads to rapid fluid loss and dehydration
What does the pertussis toxin do?
Pertussis toxin acts in the reverse manner to cholera for the same effect, inhibiting Gαi to increase cAMP production in lung epithelia
Describe the action of receptor tyrosine kinases (RTKs)
They are found across the membrane in an inactive state. A signal molecule in the form of a dimer then binds, stimulating kinase activity. As an active receptor tyrosine kinase they bind phosphorylated tyrosine. Intracellular signalling proteins then bind to phosphorylated tyrosines.
Thus, signal is relayed by activating signalling proteins into the cells interior
e.g. Insulin-like growth factors activate RTKs to control cell proliferation
How long do GPCRs take to have an effect?
Seconds
How long do Kinase linked receptors take to have an effect?
Hours
What are receptor tyrosine kinases an example of?
Kinase linked receptors
What are the roles a kinase linked receptor can fulfil?
Enzyme-linked receptors
Maybe more idk
What do Ras proteins do?
Regulate cellular processes: proliferation, cytoskeletal dynamics, membrane trafficking/vesicular transport
How do Ras proteins do so goddamn much?
Ras superfamily contains over 100 members: Rho, Rap, Rab, Arf etc.
How are Ras proteins activated?
Receptor Tyrosine Kinases activate Ras
(Activates adaptor protein which activates Ras-activating protein which replaces GDP bound to Ras with GTP thus activating it and continuing the signal)
What are Ras proteins?
GTPases
Discuss GTPases and disease
Damage to these 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.
What do Ras proteins activate?
the MAPK (mitogen-activated protein kinase) pathway
what does the MAPK (mitogen-activated protein kinase) pathway cause?
Changes in protein activity
Changes in gene expression
Discuss EGFR pathway and cancer
EGFR (epidermal growth factor receptor) is a RTK activated by TGFα (Transforming growth factor alpha)
The receptor activates Ras via Grb2/SOS proteins
Ras activates Raf (kinase), which in turn stimulates gene transcription via other kinases (MEK and ERK).
Mutations in EGFR, Ras and Raf are associated with tumorigenesis
These cause overexpression and/or hyperactivation of the respective proteins
What effect does the EGFR receptor pathway have on Ras proteins?
The receptor activates Ras via Grb2/SOS proteins
What is the EGDR?
epidermal growth factor receptor
Do signalling pathways interconnect?
Hell to the YES
Why med?
Fuck else to do
