Topic 5 - Cell Signalling Flashcards
1
Q
What are the components of a the general cellular communication pathways
A
- extracellular signal molecule (remains outside the cell)
- bound receptor
- intracellular signalling proteins (cascade activated)
- effector proteins
2
Q
What are three outcomes from the effector proteins
A
- altered metabolism
- altered gene expression
- altered shape or movement of cell
3
Q
What are GAP junctions?
A
- inter membrane narrow water filled channels directly connecting adjacent cells
- symmetrically exchange inorganic ions, small water soluble molecules
- no large proteins can be exchanged
4
Q
What is the structure of GAP junctions>
A
- 6 connexins (homomeric or heteromeric)
- 2 sets connect a cell with another cell
5
Q
Signal molecules can be?
A
- proteins
- peptides
- amino acids
- nucleotides
- steroids
- retinoids
- fatty acid derivatives
- gases
6
Q
These signal molecules can be released into extracellular space by… (3)
A
- Exocytosis
- Diffusion
- attached to a protein on a extracellular surface
7
Q
How do cells respond to signal molecules?
A
- selective reaction since signal always work in combination
- each cell type has a set of receptors that respond to a combination of signal
8
Q
What is the advantage of having cell signal combinations?
A
- allows for variety and desired effects
9
Q
What are the two types of receptors?
A
- cell-surface receptors
- intracellular receptors in two classes
1) nuclear receptor family
2) primary & secondary transcriptional response
10
Q
How do you ensure a cell responds specifically?
A
- proper receptor
- proper combination of signal
- has correct internal response proteins
11
Q
What are the main features of an inactive nuclear receptor? (3 domains)
A
- inhibitory protein bound to receptor
- exposed ligand binding domain
- DNA binding domain
- transcription activating domains
12
Q
How is a nuclear receptor activated to what effect?
A
- ligand binds to the ligand binding domain
- the inhibitory protein falls off and is replaced by a coactivator protein binding over the ligand binding domain & the transcription-activating domain
- the DNA binding domain binds to the receptor binding element on DNA
- transcription of target DNA occurs
13
Q
what are orphan nuclear receptors?
A
- we are unaware of the ligand that binds the receptor
14
Q
What are signal transducers
A
- protein receptors on the extracellular surface convert the extracellular ligand binding into intracellular signals - this has a direct effect on cell behavior
15
Q
What are 3 examples of cell surface receptors?
A
- GPCR coupled to G proteins
- Ion Channel Coupled Receptors (ex neurotransmitters and their change in [ ])
- Enzyme coupled Receptor
16
Q
Quickly explain Enzyme coupled receptors?
A
- receptor binds a signal outside
- receptor has a catalytic domain activated by this signal
- an enzyme associates on the intracellular side with further transduction occurring on the inside of the cell
17
Q
What are the 4 types of intercellular signalling?
A
- Contact dependent
- Paracrine
- Endocrine
- Synaptic
18
Q
What is unique about Contact-Dependent Signalling?
A
- signal is attached to an external signal surface (plasma membrane)
- on an opposite cell there is a receptor that binds the fixed signal
19
Q
What are 4 key points about Autocrine Signalling
A
- there should be rapid uptake by the target cell
- the signal should be destroyed by the ECM
- Signal should be blocked by the ECM
- antagonists will try to block this activity
20
Q
Explain what is key about Synaptic Signalling?
A
- it is extremely fast
- release of high local [ ] of neurotransmitter
- ex. acetylcholine
21
Q
How does Endocrine Signalling work?
A
- through slow diffusion of the signal
- ex. release of hormones throughout the body
22
Q
What are two pathways a cell have to reacting to a signal?
A
- FAST pathway: where there is a direct alteration to the function of the current protein in a cell (ex. phosphorylation - activation of GTPases) - directly changing cellular behavior
- SLOW pathway: where the signal activates gene expression of new proteins where this synthesis of new proteins lead to altered cell behavior
23
Q
What are the two responses in the SLOW reaction pathway?
A
1) Primary response where the signal activates gene expression of a ex. Transcription Factor
2) Secondary response where the TF changes gene expression of an effector protein now
24
Q
How do different cells have different responses to the SAME signal
A
ex. acetylcholine in skeletal muscle (contraction), heart pacemaker cells (decreased firing rate) and salivary glands (secretion)
1. each cell has different receptors
2. these different receptors are linked to different intracellular signalling proteins
3. these different intracellular signaing proteins activating different effector proteins
4. these different receptors activating different intracellular signalling proteins activating different effector proteins will activate different genes
25
Expand on the same signal different responses of different cells point
Each cell may have different concentrations of inhibitors and inducers during development which will alter the cells ability to respond to the signal
26
What are small intracellular mediators/second messengers
- will be generated in large numbers after receptor activation (ex. cAMP)
- diffuse away from the source, spreading the signal
27
What are large intracellular signalling proteins?
- these generate small intracellular meadiators (G protein eventually activating cAMP)
- they activate the next signalling/effector protein
28
What are different types of signalling pathways in a hypothetical pathway?
- primary transduction @ the membrane
- signal is relayed
- signal is tranduced and amplified
- integration
- spread
- anchor
- modulate
- effector protein activation
- Gene transcription
29
What are two ways molecular switches work?
- phosphorylation
| - GTP-binding
30
How does phosphorylation activate or inactivate molecular switches
- an inactive signal is phosphorylated by ATP and this turned ON and active
- an active signal has a phosphate removed from ATP and this turned OFF the protein
31
How does GTP-binding affect molecular switches?
- GTP binding activates the signal, while GTP hydrolysis removes a phosphate of GTP to GDP and it is inactive
- these may include heterotrimeric or monomeric GTP binding proteins
32
What are scaffold proteins?
- these proteins are bound to the extracellular receptor that when active will bind intracellular signalling proteins in ORDER and activate them
33
What is unique about Scaffold Proteins
- this process avoids cross-talk and increases specificity
| - close proximity increases specificity
34
How do these signalling complexes form (Scaffold proteins)
- an inactive receptor an inactive intracellular signalling proteins cannot interact to send a downstream signal
- when the receptor binds a ligand it activates the receptor which phosphorylated in intracellular component - this allows binding sites for each of the intracellular signalling proteins to bind in a scaffold and become activated
35
What are transient signalling complexes? - how is it controlled?
- refers to the quick formation and disassembly of these signalling complexes
- it requires to formation of the receptor when a signal comes and it becomes phosphorylated
- SO the scaffold will only form when required
36
How does a receptor interact with phosphoinoitide molecules and intracellular signalling proteins?
- the inactive receptor will bind a ligand and become activated
- the active receptor will interact with membrane bound phosphoinositides by PHOSPHORYLATING them - activating them
- intracellular signalling proteins can now bind these hyperphosphorylated phosphoinositides forming downstream signals
37
What sort of responses can these Switch-like proteins take
- either a sigmoidal response (gradually more and more response occurs)
- or ALL-or-NONE response (yes-no response) - so as more signal comes more cells will be activated
38
A sigmoidal response to switch-like receptors can have two outcomes?
- all cells may gradually become more responsive as the concentration of the signal increases
- ALL or NONE where one cell becomes fully activated activating the one beside it and so forth
39
How can cells desensitize themselves from signals? (5)
- receptor sequestration (removing the signal)
- receptor down-regulation (degrading the whole complex)
- receptor inactivation
- inactivation of the signalling protein
- produce an inhibitory protein
40
What is desensitization?
- the prolonged exposure to a stimulus may decrease the cells response
- CELLS RESPOND TO CHANGES IN SIGNAL CONCENTRATION
41
What are GPCRs?
- a 7 transmembrane protein called G-protein coupled receptors
- Ligand binds on the extracellular side causing rearrangements of an inactive G-protein and enzyme
42
What is the difference between primary and secondary messengers?
- Primary molecules typically bind outside the cell to a receptor (can be: proteins, peptides, amino acids, nucleotides, steroids, retinoids, fatty acid derivatives, gases)
- Secondary messengers activate intracellular signalling pathways, ex. cAMP
43
What is the structure of the G-protein? and what is a G-protein
consists of 3 subunits: Ras domain (alpha), beta and gamma domain all membrane bound
- a heterotrimeric GTPase
44
How do you activate the G-protein
- GPCR binds a signal which causes a conformational change and directs it to interact with the G protein
- an inactive G protein has a GDP bound in its AH domain but when the GPCR interact with it, the GDP is removed and a GTP binds and activates the Ras subunit & the Beta/alpha subunit is active now as well separately
- the active Ras can now have effector activation
45
Once the G protein is active what happens next? (Ras)
- the active Ras will activate adenyl cyclase enzyme which will convert ATP into cAMP
- cAMP will act as the first signalling molecule inside the cell to convey the signal (hence second messenger)
46
How do you increase cAMP concentration inside a cell?
- since cAMP levels are typically low in the cell it requires RAPID SYNTHESIS and RAPID BREAKDOWN of cAMP to work at adenyl cyclase threshold
- thus adenyl cyclase must have an increased activity
47
What does adenyl cyclase have to do to maintain this increase of [cAMP] in the cell?
- must outwork the degradation of cAMP by forming more cAMP
| - outcompete the cAMP phosphodiesterase
48
What are kinases capable of?
- phosphorylating other things
49
Explain from G-protein activation down to activated PKA and what it is?
- the activated G protein will activate the adenyl cyclase
- adenyl cyclase will increase the cAMP concentration in a cell
- PKA (protein kinase A) consists of 4 subunits; to regulatory subunits and 2 catalytic subunits
- the cAMP will bind to the regulatory subunits allowing the dissociation of the catalytic subunits toe become active PKA
- active PKA will be able phosphorylate transcription factors to become active and eventually affect gene expression
50
Explain what happens after the PKA is activated
- the active PKA will be able to enter the nucleus where it searches for an inactive CREB protein (cAMP Response Element Binding Protein - CRE-binding protein) and phosphorylating it
- the active CREB protein will be able to have CBP-binding protein (CREB-binding protein) to bind to it
- once active CREB and CBP are bound they will be able to bind to CRE (cAMP response element) which will activate the target gene and change the gene transcription
51
Explain what occurs when the active G protein instead activates phospholipase C (PLC)
- the active phospholipase C will target membrane bound phosphoinostide bisphosphate - PI(4,5)P2
- PLC will cleave PI(4,5)P2 into two components: diacylglycerol still membrane bound & inositol (1,4,5) triphosphate (IP3)
52
What is the function of diacylglycerol and IP3
- diacylglycerol will active protein kinase C (PKC)
- IP3 will activate PKC and also bind to Ca2+ channels on the ER to release Ca2+ on the inside which help in activating PKC
53
What does free Ca2+ in they cytosol do?
- it functions as an intracellular mediator and cause an increase in[Ca2+]
- this triggers contraction in muscle cells or secretion in secretory cells
54
What does cAMP and IP3 have in common?
- they both are second messengers as they both are the first molecules to progress the signal inside the cell
55
What is calmodulin and what is its function?
- a Ca2+ binding protein with 4 binding sites
| - binding Ca2+ induces a conformational change
56
What is one key point about Ca2+ function in a cell
- free Ca2+ has no enzymatic function but instead will activate numerous proteins and activate them
57
What is CaM-Kinase II and its function?
- Calmodulin Kinase II
| - involved as a molecular memory device
58
What is an example of G-protein directly regulating ion channels?
-
| - ex. acetylcholine reduces rate and strength of heart muscle contraction
59
What is an example of G-protein directly and indirectly regulating ion channels?
- the activated G protein activate after binding a receptor, instead the Ras component will inhibit adenyl cyclase activity while the beta/gamma subunit will open the ion channels
- ex. acetylcholine reduces rate and strength of heart muscle contraction
- ex olfactory channels indirectly by activating adenyol cyclase and cAMP, where cAMP will interact with the ion channel
60
What are three ways of desensitizing GPCRs?
- receptor inactivation
- receptor sequestration
- receptor down regulation (ARRESTIN)
61
Do GPCRs and Enzyme-coupled Receptors activate different signalling pathways?
NO
62
How can binding of ligand activate the kinase domain?
dimerization of the receptor and transphosphorylation of the other receptor
63
What are RTKs?
- a monomer which consist of a extracellular receptor, transmembrane alpha helix, and a signalling relay component made up of tyrosine wholly
- with a kinase where it is phosphorylated to have scaffold proteins to bind in this area
64
How do RTKs work?
- a signal will bind to a receptor binding
- when the signal is bound to two monomers will form a dimer (dimerization of the receptor)
- dimerization will be sealed by TRANS AUTOphosphorylation of itself
- this will eventually cause the phosphorylation along the intracellular components of RTK which will now function as BINDING sites for signalling proteins
- signalling proteins will bind in these active binding sites via a scaffold system which will relay the signal through secondary messengers
65
How does a EGF receptor kinase differ from a RTK?
- there is NO indication of trans-autophosphorylation
- first EGF will bind to two separate monomers
- these two monomers will come together and interlink where one monomer (ACTIVATOR) pushes against the other monomer (RECEIVER)
- the receiver will then phosphorylate BOTH monomers tails
66
What is different about insulin receptor?
- it is already a tetramer and does not require dimerization
67
How does insulin receptor activate the intracellular signalling proteins?
- once a signal binds to the receptor, transmembrane rearrangement will phosphorylate the tail
- this phosphorylation on the tail will form a binding site for the MEMBRANE BOUND IRS1 docking protein
68
What is IRS1? that binds the insulin receptor?
- a membrane bound docking protein that binds to the phosphorylated tail of the insulin recepor
- it is a phosphoinositide
69
What occurs to the insulin receptor after IRS1 binds to the tail?
- the insulin receptor tail will then phosphorylate the IRS1 tail at multiple sites
- these sites will serve as binding sites for adapter proteins and scaffold signalling proteins
70
What is unique about the phosphorylation sites on the tail of the RTK?
- enzymes that bind to these phosphorylated sites recognize specifically where on the tyrosine tail they are phosphorylated
- this directs the enzymes and in what order they will bind and become activated
71
How does a monomeric Ras-GTPase interact with a RTK?
- the activated RTK will bind to an adapter protein (Grb2) which will be able to bind and activae a Ras-GEF (aka SOS protein)
- this Ras-GEF functions by removing the GDP on the membrane bound Ras protein and replacing it with a GTP
- waking up the Ras-GTP beast to activate down stream signals
72
How is it possible to identify and observe the activation of the monomeric Ras-GTPase
- through FRET (fluoresnce resonance energy transfer)
- with YFP bound to Ras on the membrane and a RFP on a GTP
- so if Ras is active the blue light shown on the Ras will fluoresce the YFP to fluoresce the RFP bound to GTP (blue light will produce orange light - NO yellow light)
73
What signal transduction pathway can active Ras-GTP initiate?
- Raf the MAP kinase kinase kinase
- which phosphorylates MAP kinase kinase
- MAP kinase kinase will phosphorylate MAP kinase
74
What will the final MAP kinase do after being activated by Ras-GTP?
- it cannot phosphorylate Protein X and Protein Y to change these proteins activity
OR
- it can phosphorylate gene regulatory protein A and gene regulatory protein B to change gene expression
75
If there can be up to 5 different MAP kinase signal transductions active how do you avoid any cross-talk?
- use specific signalling molecules which bind to different receptors (ex. GPCR)
76
What can RTK and GPCRs both activate?
phosphoinositide 3-kinase (PI3K)
| - this kinase will form docking sites for intracellular signalling molecules it phosphorylates
77
What is an example where a RTK and PI3Kinase inhibit apoptosis?
- a survival signal will activate a RTK
- this RTK will activate a PI3Kinase which will phosphorylate a membrane bound phosphoinositide(4,5) bisphosphate (PI(4,5)P2) -- which forms a membrane bound PI(3,4,5)P3
- these PI(3,4,5)P3 form docking sites for PDK1 and mTOR to phosphorylate a AKt and activate it
- the active AKt will phosphoryate Bad and inactivate it
- Bad will release the inactive apoptosis inhibitory protein which becomes active
- this active apoptosis inhibitory protein is released and BLOCKS apoptosis
78
What is one unique step about cytokine receptors?
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Janus kinase (JAK)
- will cross phosphorylate each other, then the receptor tail for the transcription factors to bind, activate, and then head to the nucleus
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