PA20292- Amanda Mackenzie lectures Flashcards
What is endocrine cell signalling?
Uses hormone transported in blood to send messages throughout the body.
What is direct cell-cell signalling?
Receptor on surface of one cell detects ligand on surface of other cell. Cells physically connect.
What is autocrine cell signalling?
Cell signals to ITSELF. the molecule released acts back on itself.
What is paracrine cell signalling?
Cell signals to a nearby neighbour by release of a molecule.
What is the pathway of cell signalling (4 stages)?
Signal, reception, transduction, response.
Basic definition of a receptor?
Receptors are proteins that bind to specific ligands/agonists.
Receptors have an affinity for the ligand.
Difference between competitive and non competitive inhibitors?
Non competitive; binds somewhere else on receptor causing a conformational change so that the ligand can no longer bind.
Competitive: competes for same binding site as ligand, stops the response.
Where are nuclear receptors located?
Inside the cell. They are intracellular. Not in nucleus but in cytoplasm.
3 domains of the nuclear receptor superfamily?
Ligand binding
DNA binding
Transcription activation (gene transcription)
What must drugs be in order to bind to nuclear receptors?
LIPID SOLUBLE. they have to cross the cell membrane to reach the nuclear receptor. ( lipophillic)
Another name for ligand gated ion channels?
Ionotropic receptors.
Where are ligand gated ion channel receptors located?
The membrane of a cell. Occur in all cell types.
2 other names for g protein coupled receptors?
7transmembrane, metabotrophic
What do g protein coupled receptors consist of?
Seven transmembrane alpha helices
Intracelluar loop (the largest loop interacts with the g protein)
3 subunits: alpha beta gamma. Forms a-B-y trimer.
Binding domain for agonist on the receptor molecule.
Binding site for GDP/ GTP on the a subunit.
What does a kinase enzyme do?
It will phosphorylate it’s substrate.
Or phosphorylase itself (auto phosphorylation)
ATP is the source of phosphate
Phosphates can be added to tyrosine threonine or serine amino acids.
4 examples of ligands for ligand gated ion channels?
NDMA, Glycine, GABA, Ach
What do mechanically gated ion channels do?
Open in response to mechanical movement of adjacent structures, respond to stretch and temperature.
How do you measure the activity of an ion channel?
Use patch clamp recordings
What are patch clamp recordings?
A patch of membrane with a single ion channel on it is monitored. The current of this ion channel is recorded using an electrode. The flickering (opening and closing) of an ion channel is detected on the recording. Allows the very small current flowing through an ion channel to be measured directly.
What is the nicotinic acetyl choline receptor directly coupled to?
It is a ligand gated ion channel directly coupled to a cation selective ion channel
How many subunits is the acetyl choline receptor made up of?
5
a,a,B,δ,y
How is the acetyl choline receptor structure determined?
By crystallography experiments
What is a heteromeric ion channel?
What is a homomeric ion channel?
Heteromeric channels are composed of several different subunits
Homomeric channels are composed of the same subunits
Is the ACh receptor heteromeric or homomeric?
Heteromeric, consists of different subunits
Where a outs does ACh bind on the receptor?
ACh binding sites are on the extra cellular portion of the receptor at the interface between the alpha and adjoining subunits.
What happens when Ach binds to the ACh receptor?
The kinked alpha helices that act as a gate swing out the way and open the channel pore.
Opening the pore allows NA+ and K+ to diffuse down the channel depolarising the membrane.
The permeability of ____ increases when Ach binds to the Ach receptor?
Na+ and K+ . This causes the membrane to depolarise carrying the action potential.
Difference between nicotinic and muscarinic Ach receptors
Nicotinic are ionotropic receptors, meaning that they allow ions to pass through them when they bind to acetylcholine. Therefore, these receptors help depolarize the cell in response to acetylcholine and are excitatory.
Muscarinic are metabotropic receptors (G-protein coupled receptors). They activate G-proteins in response to acetylcholine and these G-proteins can have different cellular responses depending on which G-proteins are activated.
What is the ultimate response of the Ach signal binding to ACh receptors?
Muscle contraction.
Opening of Na+ channel (reception) then Influx of Na+ and depolarisation of membrane (transduction) results in Ca2+ release which in turn results in muscle contraction as the ultimate response.
What is long QT syndrome? What channel tends to be effected by this?
hERG (Kv1.11) is a voltage gated potassium channel.
This channel mediates the repolarisation of the cardiac action potential in the heart and is essential in keeping a normal cardiac rhythm.
Mutations in this ion channel may inhibit it causing delayed repolarisation of the heart after a heart beat. This can lead to TDP, an irregular heartbeat. This results in the prolongation of the QT interval and can result in sudden death.
Why is negative feedback in cell signalling important?
If you can’t turn a signal off it may result in a tumour forming.
With GPCRs, what happens when an an agonist binds? (explain mechanism)
GPCR undergoes a conformational change.
Causes it to have Hugh affinity for a subunit of G protein.
These bind, GDP release and GTP takes its place.
B-y subunits dissociate from a-GTP complex.
These are now active.
a-GTP complex can bind to target protein, activating it.
GTP hydrolysed to GDP by GTPase activity of alpha subunit, caused by it being attached to the target protein.
Alpha subunit now is associated with GDP again, detaches from target protein as its now inactivated.
What does the Gs subunit do to adenylyl cylase?
Stimulates it, increasing cAMP formation.
What does the Gi subunit do to adenylyl cylase ?
Inhibits it.
Decreasing cAMP formation.
Which G protein subunits are the ephindrine and acetylcholine receptors coupled to?
Ephindrine= Gs, increasing cAMP
Acetylcholine (muscarinic) = Gi, decreasing cAMP
Is the N terminal domain intracellular or extracellular? What is it for? What is the C terminal for?
N terminal is extracellular is extracellular and is the binding domain where ligands bind. It contains carbohydrates also for increased specificity.
C terminal is intracellular, it is the part that interacts with the alpha subunit of the g protein.
What do GTP/GDP do to the alpha subunit?
GTP ACTIVATES IT
GDP DEACTIVATES IT
Which G alpha subunits target adenylate cyclase?
Which target phospho lipase C?
Adenylate cyclase=Gi and Gs
PLC= Gq
What happens when cAmP binds to protein kinase A’s?
cAMP attach to the regulatory subunits of the PKA. This causes the 2 catalytic subunits to become activated and are released from the regulatory subunits. The catalytic subunits have kinase activity.(go on the phosphorylase proteins)
Which 3 amino acids present of proteins can phosphates be added to by Kinase enzymes?
Tyrosine, threonine, serine
What are CRE’s?
cAMP response elements.
They are DNA sequences that the cellular transcription factor CREB binds to to upregulate or down regulate transcription of the genes.
What is CREB responsible for?
Regulating gene transcription and is involved with nuclear signalling.
It’s a PKA target and is a cAMP response element binding protein (CRE binding protein)
How do you switch on/off phosphorylation signals?
Switch things ON using PKAs to phosphorylate things= activates it
Switch off using protein phosphatase 1, an enzyme which cleaves the phosphate group off = deactivates it.
How do you form AMP from cAMP?
Use enzyme cyclic AMP phosphodiesterase. It breaks the cyclic part of the molecule (doesn’t add or remove anything just breaks)
How many phosphate groups does adenylyl cyclase remove from ATP?
2 , forming a mono phosphate cAMP
What is the ultimate response the inositol phospholipid pathway leads to? And the CREB/PKA pathway?
IP3 pathway leads to an intracellular rise in CA2+ which leads to the response of cell proliferation, survival and death.
CREB pathway leads to a increase in gene transcription
Target molecule of the Gprotein in the CREB pathway? And the IP3 pathway?
cREB= adenylyl cyclase iP3= phospho lipase C
IP3 is short for…
Inositol 1,4,5 triphosphate
How is the IP3 signal terminated?
By phosphatases (enzymes) or by further phosphorylation forming inositol 1,3,4,5 tetrakisphosphate.
What does DAG activate?
Activates serine/threonine kinases, especially protein kinase C (in the Inositol phospholipid pathway).
Increase in cytosolic Calcium causes DAG to bind to the protein kinase C, activating it.
PKC needs to bind to DAG, calcium and phosphotide serine (a lipid) to become activated!!
What does phospho Kinase C need to bind to to become activated?
DAG, calcium and phosphotide serine (a lipid). It becomes attached to the membrane due to DAG and phosphotide
It is inactive when the pseudo substrate domain is still in tact.
Difference between receptor protein kinases and receptor LINKED protein kinases?
Receptor protein kinases;
The cytoplasmic portion of the receptor contains a tyrosine kinase. (receptor itself contains the kinase). The kinase is activated when ligands bind to receptor portion.
Receptor linked protein kinases; no natural kinase activity. When a ligand bind receptor activates an associated cytoplasmic tyrosine kinase.
What are insulin receptors and EGF receptors an example of?
Receptor protein kinases
What are integrins and cytokine receptors an example of?
Receptor linked protein kinases
What receptor protein kinase is Ras linked up to?
EGF receptor
What is Grb2?
An adaptor protein. Its SH2 domain binds to the phospho tyrosine residues of the EGF receptor. It’s SH3 domains then recruit SOS protein which in turn activates Ras by GDP/ GTP exchange.
Is Ras attached to the cell membrane? By what
Yes by a covalently attached lipid
What are low molecular weight g proteins? Example?
Superfamily of small g proteins.
They are classes as g proteins as the bind guanine nucleotides (GDP, GTP).
They’re a different class to hetertrimeric G proteins, only contain one kind if subunit.
Example is Ras.
How is Ras’s activity terminated?
By GTPase activating protein
GTPas is activated and take a phosphate group off GTP attached to Ras, converting it back to GDP and deactivating Ras.
What switches Ras on and off?
GEF (SOS protein) switches ON
GAP (activating GTPase) switches it OFF
Job of Ras?
Cell growth regulation via serine-threonine protein kinases. Activates a phosphorylation cascade.
What is the job of Rho?
Reorganises the cyto skeleton by serine threonine protein kinases. RhO ReOrganises!
What is the job of Arf?
Regulates vesicular trafficking and activated phospholipase D. Remember Arf= vesiculARf trARFficking!!!!
What is the job of Raf?
It has a role in secretory and endocytotic pathways. Just remember this.
What is the job of Ran?
Transport of RNA and proteins into and out of nucleus. Remember RAN= RNA. Transport = run/ ran.
What factors activate rho?
Soluble factors of blood serum such as lyso-phosphatidic acid(LPA).
Rho drives formation of stress fibres in cell reorganising the cyto skeleton.
How does Arf regulate vesicular traffic?
Recruiting COAT proteins, regulating phospholipid metabolism, modulating structure of actin at membrane surfaces.
Arf starts off inactive, then GDF/ GEF exchange. GTP- ARF complex binds coatomer proteins around Golgi membranes.
This forms a vesicle. When the vesicle reaches the membrane to fuse with it, coatomer proteins and ARF-GTP complexes detach.
How do proteins/ RNA pass through nuclear pores? What helps them?
Pass through with help of Ran-GDP complex and by bonding to IMPORTIN a transport protein.
The proteins contain an amino acid sequence called the nuclear localization signal.
In the nucleus, Ran becomes GTP-Ran which helps importins and proteins dissociate from eachother.
Exportins help move things out of the nucleus.
Where does Ran–> GDP Ran occur?
And Ran–> GTP Ran?
Ran combines with GDP IN THE CYTOPLASM OUTSIDE THE NUCLEUS.
Ran combines with GTP IN THE NUCLEUS.
What are the 3 steps in the MAPK pathway after Ras activation?
Activates Ras recruits and activates Raf
Raf then phosphorylates and activates MEK
MEK then phosphorylates and activates MAPK (ERK)
What does MAPK stand for?
Mitogen active protein kinase
This is a family of serine threonine protein kinases
What is HER2?
A receptor in the EGFR family.
Over expressed in breast cancers; causes cell proliferation and growth to span out of control.
HER 2 initiates the MAPK pathway
Treated with Herceptin
What is used to treat HER2 associated cancer ?
Herceptin (monoclonal antibody treatment) interferes with the HER2 receptor.
What is the Erolitnib tyrosine kinase inhibitor used to treat?
Non small cell lung cancer (NSCLC)
This drug inhibits tyrosine kinase enzymes so stops phosphorylation and therefore stops the pathway leading to cell growth and proliferation from working (MAPK pathway)!!!
What are the dangers of mutated Ras protein?
Can slow down GTP hydrolysis resulting in Ras being ‘locked ‘ in the on position.
Can cause uncontrolled cell division leading to cancer.
Mutated Ras found in human oncogenes (20-25%)
Ras is a target for anitcancer drugs
What are the 2 phosphoinositide3-kinases (PI3Ks)??
Pi-4 kinase
PIP-5 kinase
they both use ATP as the phosphate source
What does PI3K’s stand for
Phosphoinositide 3 kinases
They ADD A PHOSPHATE GROUP onto inositol phospholipids
What are PI3K’s activated by?
By receptor tyrosine kinases (RTKs) and G protein coupled receptors
Class 1PI3K’s are activated by…….class 1A PI3K’s are activated by……..and class 1Bs………
Class 1: cell surface receptors
Class 1A: receptor tyrosine kinases
Class 1B: regulated by G protein coupled receptors, act on the Gby subunit of GPCRs
What do class I PI3ks generate?
PI3s
What are class 1A PI3Ks composed of? What subunits?
P85 regulatory subunit (with 2 SH2 domains)
P110 catalytic subunit for 3 hydroxyl phosphorylation
This is a heterodimer
What are class 1B PI3Ks composed of? What subunits?
P101 regulatory subunit that interacts with the Gby subunit of GPCRs.
P110y catalytic subunit for 3 hydroxyl phosphorylation
This is a heterodimer
Which parts of class 1A and 1B PI3Ks interact with the areas in which they act on?
The REGULATORY SUBUNITS.
SH2 domains and Gby subunits attach here
What is the code for the catalytic domain in both class 1A and 1B PI3Ks?
p110!!!!!
This adds a phosphate group onto inositol phospholipids by 3 hydroxyl phosphorylation
What is the class II PI3Ks structure like?
A single p110- like catalytic subunit
Called either PI3KC2alpha ,PI3KC2beta. ,PI3KC2gamma
What are class II PI3Ks activated by?
Receptor tyrosine kinases
Cytokine receptors
Integrins
What’s the catalytic domain of class III PI3Ks called?
VPS34
How many amino acids is the pleckstrin homology domain made of?
120
This domain allows docking of proteins to plasmamembranes
When Bad is active , what does it encourage?
A cell to kill itself by apoptosis
How is Bad deactivated and what does this allow?
By protein kinase B (AKT).
It phosphorylates BAD, making it release the death inhibitory protein it was holding onto.
Promotes cell survival as Bad encourages apoptosis of a cell.
AKT also activates Tor, a protein that stimulates cells to grow in size
What does Tor do?
It’s a large serine/threonine kinase activated by AKT.
It stimulates cells to grow by enhancing protein synthesis.
What turns pI3K signals off?
Lipid phosphatase PTEN protein.
Converts PIP3 to PIP2
No presence of PiP3 means AKT is no activated and no cell growth and proliferation occurs.
It’s a tumour suppressor.
What is AKT/PKB activated by?
PIP3
Produced from activated PI3Ks
What PI3K inhibitor drugs exist to prevent cancer?
Penicillin wortmannin- irreversibly binds
LY294002- reversibly binds
What are the class 1 PI3K inhibitor drugs in clinical trials likely to treat?
3/4 treat advanced solid tumours; exelixis, GSK, Genentech
Novartis treats phase 1-11 solid tumours
How many cells are Eliminated from the body everyday?
10 billion cells.
Shedding intestinal lining,
Dying neutrophils,
Turnover of tissues
What effect does ischemia reperfusion have on a cell?
Ischemia reperfusion= lack of oxygen/ glucose to the cell
Causes membrane channels to open and extracellular fluid to flow into cell
Causes organelles to swell
Cell eventually bursts
What is the name of the enzyme that activates inactive pro caspase to caspase?
Initiator protease enzymes
Cleaves pro caspase at 2 sites
Release of the prodomain activates the caspase
How many caspases are there?
Approx. 9
What is extrinsic apoptosis mediated by?
Death receptors
Not part of cells nature
What family do death receptors belong to?
Tumour necrosis factor receptor family (TNFR)
Names of receptors:
TNFR1
CD95
Where is the death domain of a death receptor?
In its cytoplasmic tail.
Example of a death receptor?
Fas receptor
Fas protein binds to a Fas ligand on a killer lymphocyte cell
What type of cell death is mediated by death receptors?
Extrinsic apoptosis
In the intrinsic pathway what does the effector caspase 3 do?
Cleaves and inactivated cellular components such as:
Cytoskeleton components eg actin
DNA repair enzymes
PKC( eventually leas to cell growth is it was active)
Activates a DNA ase, cleaves DNA
Leads to cell death!
What do Bad and Bax have in common?
Both promote cell death
What does Bax do?
Promotes cell death by inducing cytochrome C release from mitochondria
What does Bcl-2 do?
Inhibits apoptosis
What does Bad do?
Binds and inhibits death- inhibiting proteins like Bcl2
When active it binds to and inactivates these death-inhibitory proteins
What is an IAP?
Inhibitor of apoptosis
Binds to some pro caspases to inhibit their activation
Binds to activated caspases to inhibit their activity
What would you treat osteoarthritis , aplastic anaemia (RBC death) , myocardial infarction (heart cell death) and stroke (brain cell death) with?
Treat with caspase inhibitors!
What receptors can activate phospho lipase C to increase cytosolic calcium?
Tyrosine kinase receptors, &
ACh binding to GpCRs
What 3 ligand gated ion channels associated with calcium can you think of?
ATP GATES P2X RECEPTORS
NDMA RECEPTORS (GLUTAMATE ION CHANNELS)
TRP CHANNELS
What doe capsaicin, menthol and mustard oil open?
TRP channels on cell membrane
Capsaicin opens TRPV1
Menthol opens TRPM8 (mint m8)
Mustard oil opens TRPA1
What can you think of that down regulates/ reduces the amount of calcium in the cytosolic?
NCX exchanger in cell membrane pumps Ca2+ out and Na+ in.
SERCA; membrane of ER/ SR. ca2+ ATPase, transfers CA2+ from cytosol into SR/ER.
PMCA; plasma membrane calcium ATPase. Removes CA2+ from the cell.
What refills calcium stores in the muscle cell?
Ca2+ ATPase called SERCA
What is the EF hand?
A helix - loop- helix structural domain found in a large family of calcium binding proteins.
Example of a member of the EF hand family?
Calmodulin
Has 4 Ca2+ binding sites in its loop domain
Targets calcium dependent protein kinases
And targets plasma membrane Ca2+ATPase pump
What is calmodulin?
A calcium binding messenger protein CARRIER OF CALCIUM
Transduces calcium signals by binding to calcium then interacting with target proteins.
As many of the proteins CaM binds to cant bind calcium themselves.
How do we detect calcium?
Fura-2 calcium indicator
Fluorescent indicator, changes colour when binds to calcium.
Membrane impermeable; add AM to make it permeable
Forms fura2AM
Once in cell, AM cleaved back off
Dye is now trapped in the cell.
Detects changes in cytosol [calcium]
What does the FGF receptor inhibitor PD173047 do?
Inhibits the fibroblast growth receptor.
Stops small cell lung cancer growth
Inhibits FGF2 signalling pathways (uses Ras as signalling molecule then MAPK pathway)
This pathways blocked, stops cell growth and proliferation
How does PKB associate to the cell membrane?
Phosphorylation of membrane lipids
