Cell Signalling Flashcards
1
Q
Define cell signalling?
A
“a complex system of communication that governs basic cellular activities and coordinates cell actions”
2
Q
What is Signal transduction?
A
The relay of information from receiving signals, to processing the information, to determining and undergoing an action
- conversion of a signal from one thing to another
3
Q
What is paracrine signalling?
A
- signals are operating over short ranges
- signalling molecules are called local mediators
- ligands have structural features that restrict their diffusibility
- important for local modifications (e.g. in wound healing)
4
Q
What is synaptic signalling?
A
- a type of paracrine signalling
- neuronal cell types
- vesicle packets released at synapses
- signalling can only occur within synapses
5
Q
What is endocrine signalling?
A
- specialised cell types that are releasing the signalling molecule into the blood stream
- made available to target cells
- long range signalling
- e.g. beta cells releasing insulin
6
Q
What is Juxtacrine - contact dependent signalling?
A
- the extracellular signal cannot diffuse
- attached to signalling cell
- must have physical contact
7
Q
What are the 3 main events that occur during cell signalling?
A
Reception
- at cell surface
Transduction
- through cytosol
Response
- doesn’t have to be in the nucleus unless it is gene expression related
- cytosol ( e.g. changes to cytoskeletal proteins)
8
Q
What are the three classes of receptors that respond to signals?
A
- ion-channel coupled receptors
- when ligand binds, conformational change occurs and ion is allowed in
- rapid signalling
- e.g. acetylcholine receptors - g-protein coupled receptors
- function by switching mechanism that involves GTP and GDP - enzyme-coupled receptors
- when ligand binds, leads to activation of a protein/enzyme catalysis process
9
Q
What is the structure of GPCR?
A
- 7 transmembrane helices
- extracellular ligand binding site
- hydrophobic pocket is created by the helices which the ligand can bind
10
Q
What happens when a ligand binds to the G-protein coupled receptor?
A
- ligand binds in hydrophobic pocket
- this elicits a transmembrane conformational change in the conformation of the helices
- intracellular signalling is initiated by sensing the conformational switch
11
Q
What is the structure of acetylcholine receptors?
A
- 2 α, β, γ, δ subunits forming the receptor
- the 2 α are acetylcholine binding
- Leucine residues from each of the subunits that block the pore
12
Q
How does the acetylcholine receptor work?
A
- binding of the ligand causes conformational change that moves the leucine residues out of the way, unblocking the pore
- negative charges in the subunits make sure that specificity is met and only charged ions pass through
- influx of Na+
- part of membrane depolarisation that causes muscle contraction
13
Q
How do enzyme coupled receptors work?
A
binding of the ligand will cause dimerisation of the receptor subunits
- causes activation of the internal catalytic domain or recruitment of an associated enzyme
14
Q
What are the different modes of activation?
A
- binding/ dissociation causing activation
- post-translational modification causing activation
- conformational change causing activation
- localisation causing activation (moving to the nucleus to become active for e.g.)
15
Q
What are the different proteins involved in molecular switches?
A
Kinases, Phosphates, GAP and GEF
16
Q
How do Kinase enzymes work?
A
enzymes that catalyse the transfer of phosphate groups from ATP to one or more Ser, Thr or Tyr residues on specific substrates
17
Q
How do Phosphatase enzymes work?
A
enzymes that remove phosphate groups from substrates
18
Q
How do GAP enzymes work?
A
- GTPase activating protien
- binds to activated GTPase stimulating the GTPase activity, causing the enzyme to hydrolyse its bounds GTP to GDP
- Results in termination of the signalling event
19
Q
How do GEF enzymes work?
A
- Guanine nucleotide exchange factor
- binds to activated GTPase, causing it to release its bounds GDP
- allowing GTP to bind
20
Q
What is phosphorylation?
A
A translational modification
- regulated by kinases and phosphates
- can lead to 3 different changes in the protein
- cause conformational change
21
Q
What are the 3 possible changes that can happen to the protein during phosphorylation?
A
Conformational Change - phosphate group is negative - negative charge can attract + charge a.a causing the change Form a recognition site for other proteins - Promote protein binding - SH2 domain Disrupt protein-protein interactions - Prevent protein binding
22
Q
What are receptor tyrosine kinases?
A
- an enzyme coupled receptor
- have intrinsic kinase activity
- different extracellular domains
- phosphorylate substrates on tyrosine residues
- ligand binding outside the cell activates the kinase inside the cell
23
Q
What is the structure of the tyrosine kinase domain?
A
- have 2 domains linked together by a flexible region
- N terminal domain contains an ATP binding site
- C terminal domain contains substrate binding site and 2 alpha helices that determine specificity by the exposed amino acids in the helix
- activation loop in the C terminal domain
- contains one or more Tyr molecules
- binds to catalytic cleft
24
Q
What 2 conformations is the activation loop in for tyrosine kinase domain?
A
unphosphorylated form - sits in the substrate binding site
phosphorylated form - the loop flips away from the binding site
25
How does the RTK become activated?
- ligand binds
- receptor dimerisation and inter-molecular auto-phosphorylation of another tyrosine kinase
- cluster together to exert kinase activity
- causes docking sites
26
What do the RTK's that are not heavily phosphorylated recruit?
Scaffold protein
| - this can be phosphorylated and can recruit the other proteins (effector proteins)
27
What are protein interactions domains?
- conserved structural units of a protein (35-150 a.a)
- will bind to different motifs
- e.g. SH2 & PTB domain binds to a RTK motif, PH domain binds to lipids
28
What is the structure of the SH2 domain?
```
2 binding sites
- binding site for the phosphotyrosine
- binding site for the side chains of flaking amino acids (that make up the motif)
pY-X-X-X-(X-X-X)
- have specificity
```
29
How does the pY-insulin receptor undergo signalling?
IRS1 PTB recognises one of the phosphorylation sites on the pY-insulin receptor
- IRS1 is recruited to the membrane
- IRS1 is phosphorylated
- The phosphorylated residues then will then form specific motifs that will recruit effector proteins
- effector proteins will transduce proteins downstream
30
What pathway does PI3Kinase activate?
Akt
31
What pathway does Grb2 and SH2 activate?
MAPK
32
What is the MAP kinase pathway?
1. Grb2 adaptor protein binds to either RTK or docking proteins and binds to the phosphates via its SH2 domains
2. SH3 domains on Grb2 recruit Sos (a GEF)
- Sos causes the release of GDP
3. Activates Ras protein recruits Raf to the membrane to be phosphorylated and activated by another kinase
- inactive GDP form is bound to the membrane
- after activation of Ras, MAPK cascade occurs
2. Raf phosphorylates Mek
3. Activated Mek phosphorylates Erk
4. Erk phosphorylates many other proteins that undergo changes
33
Why is the MAP kinase pathway important?
- mitogenic
- regulate mitosis, cell division and proliferation
- associated with Cancer
34
What are the targets for Erk (MAP kinase)?
Nuclear
- can cause changes in gene expression in the nucleus
- Myc: drives the cell through the cell cycle (cell proliferation)
Cytoplasmic
- can interact and activate proteins in the cytoplasm
35
What is the G protein activated pathway (alpha subunit)?
- ligand binding = adrenaline
1. starts off with an activated α subunit of G protein
2. activates membrane bound enzyme adenyl cyclase
3. adenyl cyclase converts ATP to cAMP
4. cAMP binds to PKA, which is then activated by the release of the inhibitory R subunit from the C subunit.
5. Translocation of PKA to the nucleus
6. Activation of CREB transcription factor
- once phosphorylated, will bind to the response element
- gene expression
36
How do you inactivate cAMP?
Using enzyme cAMP phosphodiesterase
37
What are the different responses that are mediated by cAMP?
```
Heart
Hormone: adrenaline
Major response: Inc in heart rate and force of contraction
Kidney
Hormone: vasopressin
Major response: water reabsorption
Muscle
Hormone: adrenaline
Major response: glycogen breakdown
Ovary
Hormone: luteinising hormone
Major response: progesterone secretion
```
38
What is the G protein activated pathway (beta and gamma subunit)?
1. starts off with an activated βγ subunit of G protein
2. activates enzyme phospholipase C
3. cleaves PIP3
- accumulation of IP3 and DAG
4. DAG recruits protein kinase C
4. IP3 acts as a ligand for a calcium channel in the ER
- will bind to the gated channel on the ER membrane and release Ca2+ into the cytosol
- Ca is needed to activate PKC
39
What is the difference between cell signalling and gene expression networks?
Cell signalling:
- enables transmission from outside of cell to nucleus
- fast ON and OFF (s to m)
- spatial/directional responses and organisation
- energetically cheap (no protein synthesis)
Gene expression:
- slow ON and OFF (m to h)
- stable changes
- limited spatial responses
- energetically costly (and translation)
40
What is desensitisation?
switching off the signalling pathways
41
What are the different mechanisms in which desensitisation occurs?
Receptor sequestration
- receptor mediated endocytosis
- receptor is engulfed into endosome inside the cell so cannot receive any signals
Receptor down-regulation
- receptor mediated endocytosis
- receptor is engulfed into a lysosome and enzymes degrade the protein
Receptor Inactivation
- inactivated via dephosphorylation by a phosphatase
Inactivation of signalling protein
- signalling protein downstream is inactivated
Production of inhibitory protein
- product produced by the signalling protein inhibits a pathway (GAP)
42
How does receptor-mediated endocytosis work?
1. receptor and ligand are on membrane (clathrin coated pit in the inside)
2. Buds off to form vesicle
3. Vesicle can either be recycled back into the membrane or fuse with a lysosome and be degraded
43
How are GPCR's desensitised?
- phosphorylated by kinase (GRK)
- the phosphorylation will recruit arrestin
- Arrestin initiates receptor mediated endocytosis
44
How are signalling events studied in the lab?
- phosphoantibodies
- perturbation of cell signalling pathways
- mutant proteins
45
How do phosphoantibodies aid us in studying cell signalling?
1. design a synthetic peptide that was phosphorylated
2. immunise a rabbit
3. collect the antibodies created by the rabbit against the peptide
4. use antibody to probe cells to look for levels of phosphorylation
- antibody will bind to the phosphorylated peptide
- won't bind to an unmodified peptide
- specific binding
46
How does perturbation of cell signalling pathways aid us in studying cell signalling?
- finding other pathways that may contribute to the outcome being studied
1. use knock-out or knock-down techniques to get rid of one of the proteins
- if the protein is inhibited, will the same outcome occur?
47
How does mutating proteins aid us in studying cell signalling?
- mutating particular amino acids
- such as knowing certain Tyr are needed in RTK and mutating to Ala
- show which proteins still bind to receptor
48
What happens when cell signalling goes wrong?
- signal not sent at the right time
- signal doesn't reach target
- target doesn't respond
- cells respond without a target
DISEASE (such as cancer)
49
How can a defective Ras protein cause cancer?
- oncogenic mutation causes Ras to be constitutively bound to GTP
- too much MAP Kinase signalling and cell proliferation
( e.g. point mutation in RasK causes pancreatic cancer)
