Cell Signalling Flashcards
(79 cards)
1
Q
What are the three parts of a cell surface receptor?
A
Extracellular domain
Transmembrane domain
Intracellular domain
2
Q
What happens with a ligand bonds to a receptor?
A
Induced confirmation changes which are important in receptor activation
3
Q
Intracellular second messengers
A
- Simple molecules that serve to amplify the stimulus
- The concentration of second messengers increases in the cytosol following cell stimulation and decreases when the stimulus is removed
- An increase in the concentration of second messengers activates the target proteins with the result that the stimulus is relayed further in the cell
4
Q
What do intracellular signalling proteins do?
A
Relay information from protein to protein and thereby transmit the signal closer to its final target. They may also amplify the signal
5
Q
Two examples of mechanisms of information relay between intracellular signalling proteins
A
- Protein-protein interactions (conformational changes)
2. Protein post-translational modifications
6
Q
What do protein kinases do?
A
Phosphorylate their substrate
7
Q
What do protein phosphatases do?
A
Dephosphorylate their substrate
8
Q
What do protein kinases and protein phosphatases function as?
A
Molecular switches - activate or deactivate their substrates
9
Q
How can protein kinases work together?
A
Cause a phosphorylation cascade
10
Q
What is chemotaxis?
A
The directed motion of an organism (in this case bacteria) towards environmental conditions it seems attractive and/or away from surroundings it finds repellent
11
Q
How do the flagella allow movement in E. coli?
A
- When the flagella rotate counterclockwise they bundle and E. coli moves forward in a smooth swimming run
- When they rotate clockwise this disrupts the flagellar bundle and the cells tumble
- Tumbles last only a fraction of a second which is sufficient to effectively randomise the direction of the next run
12
Q
What is the tumble rate in a homogeneous environment?
A
1 per second
13
Q
How must the tumble rate change to move towards an attractant?
A
Decrease tumble frequency and increase bias in favour of smooth runs
14
Q
What is FliM?
A
The switch controlling the direction of rotation
15
Q
What are the two components of a signalling system (at its most simplified)?
A
- A histidine protein kinase (HPK) / sensor kinase
2. A response regulator (RR)
16
Q
What are the names of the two components of the bacterial chemotaxis signalling system?
A
1: HPK = CheA
2. RR = CheY
17
Q
Two domains of the HPK (histidine protein kinase)
A
Input domain
Transmitter domain
18
Q
Two domains of the RR (response regulator)
A
Receiver domain
Output domain
19
Q
General process by which response is generated in signalling system
A
- HPK input domain senses the stimulus
- Input domain activated the transmitter domain
- Transmitter domain has protein kinase activity, and autophosphorylates on the histidine residue
- The phosphate from the phosphohistidine residue is transferred to an aspartate residue on the receiver domain of the RR (known as phosphorelay)
- This indices a conformational change in the output domain of the RR
- The output domain generates a response
20
Q
Cell signalling process for bacteria chemotaxis
A
- Repellent detected by MCP dimer
- MCP activates CheA leading to trans-autophosphorylation on histidine residue
- The phosphate is transferred to aspartate on CheY
- CheY-P interacts with FliM
- Motor rotates clockwise
- Tumble
21
Q
Switching off cell signalling that causes tumble in bacterial chemotaxis
A
- CheZ is a phosphoprotein phosphatase that is activated by increased concentrations of CheY-P
- Once activated, it dephosphorylates CheY-P
- This stops the interaction with the FliM so the motor turns anticlockwise
22
Q
What does CCK stand for?
A
Cholecystokinin
23
Q
What does GPCR stand for?
A
G protein coupled receptor
24
Q
What is CCK?
A
A polypeptide secreted by the mucosal cells of the duodenum into the blood stream in response to the presence of the products of the digestion of foods
25
What happens when CCK reaches the pancreas?
It binds to its GPCR (present as a dimer) on the surface of pancreatic acinar cells causing the secretion of alpha-amylase into the bile duct, which empties into the duodenum
26
What does alpha-amylase do?
Breaks down starches during digestion
27
What do G proteins bind?
Guanosine diphosphate (GDP) and guanosine triphosphate (GTP)
28
Features of G proteins which interact with G protein coupled receptors
- G proteins are heterotrimeric (composer of three subunits - alpha, beta and gamma)
- The alpha subunit has GTPase activity
- G proteins act as molecular switches, molecular timers and amplifiers
29
How does a G protein act as a molecular switch?
Activated signal transduction cascade when GTP is bound
30
How does a G protein act as a molecular timer?
Due to GTPase activity
31
How do G proteins act as amplifiers?
Interact with multiple copies of the effector protein
32
What does the G protein alpha subunit activate?
Phospholipase C (PLC)
33
Process of G protein coupled receptors interacting with G proteins
1. Binding of hormone induces a conformational change in receptor
2. Activated receptor binds to G(a) subunit
3. Binding induces a conformational change in G(a); bound GDP dissociates and is replaced by GTP; G(a) dissociates from G(B/Y)
4. Hormone dissociates from receptor; G(a) binge you effector, activating it
5. Hydrolysis of GTP to GDP causes G(a) to dissociate from effector and reassociate with G(B/Y)
34
What happens when phospholipase C is activated?
PLC hydrolyses a plasma membrane phosphoglyceride known as PIP2. The products of this reaction are the intracellular second messengers IP3 and DAG
35
What does IP3 do once produced?
IP3 is water soluble and diffuses into the cytosol where it binds to its receptor on the ER
This results in an increase in the calcium concentration of the cytosol [Ca2+]cyt
36
What happens when the concentration of calcium in the cytosol increases due to the action of IP3?
This causes PKC (protein kinase C) to migrate to the plasma membrane where it is activated by DAG. Activated PKC participates in alpha amylase secretion
37
How to switch the signal off and stop alpha amylase secretion?
[Ca2+]cyt is returned to its pre-stimulus (resting) concentration through the action of systems such as Ca2+-ATP-ase enzymes that pump Ca2+ back into the endoplasmic reticulum or out of the cell
38
How do acinar cells have polarity?
The zymogen granules are located in the apical region close to their site of secretion
39
What do zymogen granules contain?
Inactive digestive enzymes alpha amylase and trypsinogen
40
What does addition of CCK to pancreatic cells cause?
Induces cytosolic calcium ion concentration to oscillate in the apical region triggering secretion
41
What can cause pancreatitis?
Caused by migrating gallstones (through bile reflux into the pancreatic duct) and excessive alcohol (non-oxidative products). These cause the release and activation of the digestive enzymes from the zymogen granules into the acinar cells. Once released the enzymes cause necrosis by digesting the cell
42
How is the rising phase of the cytosolic [Ca2+] oscillation achieved?
The rising phase is achieved through the operation of ‘on mechanisms’ (both direct and indirect)
43
Direct ‘on mechanisms’
The involvement of plasma membrane calcium permeable ion channels
- Voltage operated calcium channels: channel opening controlled by plasma membrane voltage
- Ligand gated calcium channels: ligand binding opens the channels
- Second messenger operated calcium channels: an increase in the concentration of an intracellular second messenger in the cytosol opens the channel
44
Indirect ‘on mechanisms’
The release of calcium from intracellular stores through the action of calcium mobilising intracellular stores
a) InsP3: generates through the activity of PLC
b) Cyclic ADP ribose (cADPR) generates through the activity of ADP ribosyl cyclase
c) Nicotinic acid adenine denucleotide phosphate (NAADP) generated through the action of ADP ribosyl cyclase
d) Sphingosine-1-phosphate (S1P) generates through the activity of sphingosine kinase
45
What is the difference between if NAD or NADP is the substrate for ADP ribosyl cyclase?
If NAD is the substrate, then cyclic ADP ribose (cADPR) is produced
If NADP is the substrate, nicotinic acid adenine denucleotide phosphate (NAADP) is produced
46
What does cADPR do?
Induces Ca2+ release from the ER, which involves the ryanodine receptor Ca2+ channel
47
What does NAADP do?
Releases Ca2+ from a lysosome-like organelle
48
Off mechanisms to prevent [Ca2+]cyt oscillation
- Na+/Ca2+ exchanger
- Plasma membrane Ca2+-ATPase (PMCA)
- Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)
49
What signals to opening of stomata?
High light
Low CO2
Leads to high turgor of guard cells
50
What signals to closing of stomata?
ABA (abscisic acid)
Calcium chloride
Leads to low turgor of guard cells
51
How does signal transduction cause stomatal closure?
The loss of guard cell turgor is achieved in part by the loss of K+ and Cl- from the guard cell through the plasma membrane K+ and Cl- ion channels to outside the cell.
Signals such as ABA and CaCl2 cause stomatal closure.
A signal transduction pathway involving receptors, PLC and protein kinases is responsible for coupling the closure-inducing stimulus to the opening of ion channels responsible for K+ and Cl- efflux from the guard cell. This pathway includes a stimulus-induced increase in [Ca2+]cyt
52
The mutant of what gene prevents stomatal closure, and what does the gene encode?
Who is paper by?
GCA2
Encodes a protein kinase
Mutant has different pattern of oscillations caused by ABA addition to wild type
Allen et al. 2001
53
What happens in the Arabidopsis det3 mutant and how does it affect stomatal closure?
Who is paper by?
There is a lesion in an enzyme that contributes to Ca2+ pumping into the vacuole
As a result external Ca2+ cannot generates oscillations in [Ca2+]cyt and does not bring about stomatal closure
Evans and Hetherington, 2001
54
How do stimulus-induced increases in [Ca2+]cyt control genre expression?
NFAT signalling pathway
NFAT is a transcription factor
It is present in the cytosol in an inactive state
It is activated in response to an increase in [Ca2+]cyt and migrated to the nucleus where it interacts with other proteins to control the expression of its targets
55
Pathway by which NFAT controls gene expression
1. An increase in Ca2+ activates a calcium binding protein known as calmodulin
2. Activated calmodulin then binds to and activates calcineurin (a protein phosphatase)
3. Calcineurin dephosphorylates NFAT and exposes its nuclear localisation signal. NFAT then shuttles into the nucleus where it interacts with other proteins and controls the expression of its target genes
4. NFAT shuffles our of the nucleus after it has been phosphorylated by the protein kinases GSK3beta or p38
56
What does NFAT stand for?
Nuclear Factor of Activated T cells
57
What does TKR stand for?
Tyrosine kinase receptor
58
Features of TKRs
- Very large family
- Involved in many responses - insulin and many growth factors operate through TKRs
- TKRs possess intrinsic tyrosine kinase activity (unlike GPCRs which have no endogenous enzymic activity)
59
Example of typical tyrosine kinase receptor (TKR)
The epidermal growth factor receptor
Single polypeptide crosses the PM once
Has intrinsic tyrosine kinase activity
60
Steps of TKR activation (steps 1-4)
1. Ligand binding sites empty, tyrosine kinase exhibits minimal activity
2. Ligand binding induces receptor dimerisation and this induces trans-autophosphorylation of activation lip tyrosines
3. Activated tyrosine kinase phosphorylates additional tyrosine residues on other regions of the TKR cytoplasmic domain and these create binding sites for additional signalling proteins
4. Recruitment of other proteins
61
Step 4 of TKR activation: recruitment of other proteins
- GRB2 binds to phosphotyrosine residues on the TKR via its SH2 domain
- Sos is recruited to the complex by binding to the GRB2 SH3 domain
- Sos then interacts with Ras
62
What is Ras?
Ras is a GTPase (hydrolyses GTP to GDP) so is another example of a G protein
However, unlike the 3 subunit-containing G proteins (that couple to GPCRs), Ras is monomeric
Ras acts as a molecular switch and molecular timer
When Ras binds GTP the switch is ON, whereas when GDP is bound the switch is OFF
63
The monomeric protein cycle (How does Ras act as a switch and timer?)
- Switching Ras on (GTP bound) is accelerated by the GEF protein
- Switching Ras off (GDP bound) is accelerated by the GAP protein
- The rate of GTP exchange serves as the timer
64
Steps 5+ of TKR activation
5) Sos is a GEF so promotes GTP bonding to Ras and hence switches it on. Ras (ON) dissociates from Sos and activates the next component in the pathway called Ras
6) Ras recruits and activates the protein kinase Raf and the 14-3-3 protein. Raf activates the protein kinase MEK. MEK activates the protein kinase MAP kinase. MAP kinase migrates to the nucleus and activates gene transcription
65
What activates and deactivates Ras?
GEF protein accelerates switching on Ras
| GAP protein accelerates switching off Ras
66
What causes 50% of colorectal cancers
Single activating point mutation in K-ras oncogene
67
How does the protein kinase MAP activate gene transcription?
- Active MAP kinase (dimer) phosphorylates the protein kinase p90 and migrates to the nucleus
- In the nucleus MAP kinase phosphorylates the TCF protein
- In the nucleus the activated p90 protein kinase phosphorylates the SRF protein
- SRF and TCF proteins bind to a sequence of DNA called SRE in genes involved in the control of cell proliferation
68
What are cytokines?
A family of small secreted proteins that control many aspects of growth and differentiation
69
What does Epo stand for and what is it?
Erythropoietin - cytokine which triggers red blood cell production
70
How does Epo work?
A drop in blood O2 is sensed by kidney cells. Kidney cells secrete Epo into the bloodstream
RBCs are derived from haematopoetic stem cells via erythroid progenitor cells
Epo binds to its receptor on the progenitor cells and inhibits apoptosis thereby promoting the production of more RBCs
71
What is the Epo receptor?
A cytokine receptor
| Single transmembrane pass protein
72
JAKs
Tyrosine protein kinases permanently associated with the cytokine receptor
73
STATS
for Signal Transducers and Activators of Transcription
74
Epo signalling steps
1. Binding of Epo to receptor causes receptor dimerisation
2. Dimerisation of receptor causes JAKs to phosphorylate each other (on tyrosine residues)
3. Phosphorylation of JAKs results in JAK activation
4. Activated JAKs phosphorylate the receptor
5. The phosphotyrosine residues create binding sites for STATs
6. STATs bind to receptor and are phosphorylated by JAKs
7. Phosphorylation of STATs causes them to dissociate from the receptor
8. Phosphorylated STATs dimerise in the cytosol then migrate to the nucleus where they control gene transcription
75
What does acetylcholine cause in blood vessels?
Causes the synthesis of nitric oxide in the endothelium which causes smooth muscle to relax
76
Who discovered that acetylcholine causes the synthesis of NO?
Robert Furchgott
77
NO signalling process
1. Acetylcholine (ACh) binds to GPCR on endothelial cell causing an increase in the Ca2+ concentration in the cytosol
2. This activates NO synthase which produces NO
3. NO diffuses out of the endothelial cell into the muscle cell
4. NO activates gyanyl cyclase (GC)
5. Gaunyl cyclase produces cGMP and activates protein kinase G (PKG)
6. This leads to the relaxation of smooth muscle
78
How does viagra work?
- In response to the appropriate nerve impulses NO is synthesised and causes the smooth muscles surrounding the blood vessels in the penis relax
- The resulting vasodilation causes the penis to become engorged with blood - erection
- Erection maintained by the engorgement of penis with blood. This is promoted by the continual presence of NO and increased levels of cGMP
- In a clinical situation erectile dysfunction wan be alleviated by preventing the breakdown of cGMP
79
What is the clinical name for viagra?
Sildenafil citrate
