1
Q
What is cell signalling the ability of the cell to do
A
- Detect or receive information
- Process the information
- Respond to generate events fundamental to living
2
Q
What does cell signalling allow for
A
- Specialist functions
- Co-ordination with other cells
3
Q
Why are signaling molecules and their receptors the main targets for theraputic drugs
A
because abnormal cell signalling underpins most disease processes
4
Q
What controls the breeding behaviour of prairie voles
A
by the action of related peptides oxytocin (females) and vasopressin (males). Act through their specific receptors found in regions of the brain concerned with mating
5
Q
What is the first principle of cell signalling
A
Cells communicate with each other via extracellular signaling molecules (also known as ‘first messengers’)
6
Q
Intracellular signalling
A
- Signaling cell produces a signaling molecule (LIGAND) - can travel short or long distances (or no distance at all)
- Signaling molecule is detected by a receptor on (or in) the target cell
- Receptor is specific for that signaling molecule – allows for control and specialized functions
7
Q
What are the 2 broad classes of extracellular signalling molecules
A
- Large and/or hydrophilic (water soluble) - bind to cell surface receptors
- Small and/or hydrophobic – enter cell and bind to intracellular receptors
8
Q
Paracrine intercellular communication
A
Released signal affects cells in close proximity (‘local mediators’). Limited travel ability. Examples: some growth factors, histamine, nitric oxide
9
Q
Autocrine intercellular communication
A
Sender and target cell are the same. Examples; molecules regulating development; some growth factors
10
Q
Endocrine intercellular communication
A
Usually, the signal acts on distant cells (but can act on nearby cells). Hormones. Examples: insulin, glucagon, testosterone, oestrogen, adrenaline (epinephrine)
11
Q
Synaptic intercellular communication
A
Axon of neurone transmits an electrical signal over long distances. At axon terminal, electrical signal causes the release of neurotransmitter messenger molecules into the synapse e.g., acetylcholine, GABA. Neurotransmitter travels short distance only to specific target cell
12
Q
Juxtacrine (or contact dependent) intercellular communication
A
The signaling cell is in direct contact with target cell
13
Q
What is the second principle of cell signaling
A
signal transduction - process whereby one type of signal is converted into another type
14
Q
What is signal transduction linked with
A
cell surface receptors
15
Q
What is the process of signal transduction
A
Begins when receptors on the cell surface receive the signal and convert or relay the ‘message’ to a molecule inside the cell. Signal is subsequently transduced along many intracellular molecules (also known collectively as ‘second messengers’) I.e., INTRACELLULAR SIGNALING
16
Q
What is the third general principle of cell signaling
A
the response of the cell can be fast or slow
17
Q
Slow v fast response of cell
A
A slow response may be protein synthesis being altered whereas a fast response may be the protein function being altered
18
Q
What is the 4th principle of cell signaling
A
The same signal molecule can induce different responses in different target cells
19
Q
How can the same signal molecule induce different responses in different target cells
A
- Variants or isoforms of the same receptor
- Similar receptors use different intracellular signal transduction pathways
20
Q
Example of same signaling molecule causing different effect
A
epinephrine beta receptor - vessel dilates. epinephrine alpha receptor - vessel constricts
21
Q
How can a cell surface receptor relay extracellular signa;s via intracellular signaling molecules
A
- Acts like molecular relay as ‘message’ is transduced from molecule to molecule
- Final molecule in sequence interacts/activates an effector protein – cellular response
22
Q
How is information transferred in the signal transduction pathway
A
by changes in the state of the protein - which is detected by the next molecule in the sequence which then becomes altered e.g., change in shape
23
Q
What can cause a protein to change shape
A
- Molecules simply binding with each other
- Addition/removal of a phosphate to the molecule
- Molecule binds to phosphate on another molecule
24
Q
What is the point of a signal transduction cascade
A
- Amplify the original signal
- Integrate and distribute signals coming from other signal transduction pathways (Note: scaffold proteins allow for some signaling components to be activated more efficiently)
25
What are signal transduction pathways comprised of
1. Proteins: including enzymes
2. Lipids: e.g., phospholipids, ceramides, diacylglycerol (DAG)
3. Small chemical mediators e.g., cAMP, cGMP, inositol triphosphate (IP3)
4. Ions: e.g., Ca2+, Zn2+
5. Gases e.g., nitric oxide
26
What can activate/deactivate signal transduction molecules
1 – binding to guanine nucleotides – GTP and GDP
2 – phosphorylation
27
What are G proteins
intracellular proteins that are regulated by binding to guanine nucleotides
28
What activates and deactivates G proteins
inactive - bound to GDP
active - bound to GTP
29
What does GTPase do
hydrolysis of GTP to GDP (switches off protein)
30
What 2 forms do G protein exist in
when trimeric complex (used by G-protein coupled receptors) and as a single monomeric protein
31
What does activation/inactivation of monomeriic G proteins require
GEFs to aid in GDP/GTP exchange and GAPs to aid in GTP hydrolysis
32
What does activation/inactivation of monomeriic G proteins require
GEFs to aid in GDP/GTP exchange and GAPs to aid in GTP hydrolysis
33
Key members of monomeric G proteins
1. Ras – cell division and growth
2. Rab – membrane transport and vesicular transport
3. Rac and Rho – cytoskeleton organization migration
34
What do protein kinases do
Add phosphate from ATP to specific amino acids on target protein e.g., tyrosine kinase and serine/threonine kinase
35
What can reverse protein kinase effect
protein phosphates
36
How are protein kinase switch proteins
by being activated/deactivated by phosphorylation
37
Once a protein kinase is activated what can it do
phosphorylate and activate the next protein kinase in the sequence
38
How is cAMP produced
from ATP by the enzyme adenyl cyclase
39
What does adenyl cyclase consist of
consists of two transmembrane domains, joined by a catalytic intracellular domain
40
What is cAMP degraded from
a cyclic nucleotide to a 5’ monophosphate (AMP) by a cAMP phosphodiesterase
41
What mediates cAMP responses
via cAMP – dependent protein kinase A I.e., Protein kinase A (PKA)
42
What does inactive PKA consist of
2 regulatory (R) subunits and 2 catalytic (C) kinase subunits
43
What does cAMP bind to
the regulatory subunits causing the molecule to dissociate. 2 resulting monomeric kinase units are active and can bind and phosphorylate target proteins
44
What is PIP2
PIP2 (phosphatidylinositol 4,5-bisphosphate). Cell membrane phospholipid. Found in inner leaflet of lipid bilayer
45
What is Phosphoinositide comprised of
hydrophobic diacylglycer5ol (DAG) lipid tall, hydrophilic inositol sugar as head group (inositol triphosphate – IP3)
46
What does P13-kinase do
phosphorylates PIP2 in the lipid bilayer to PIP3
47
What is the key regulatory molecules in the P13-K pathway
PTEN which dephosphorylates PIP3 back to PIP2 which shuts down the signaling via PKB
48
what is PDK1
phosphoinositol-dependent kinase - binds to PIP3 which can activate Akt
49
What breaksdown PIP2 in the lipid bilayer
phospholipase C (PLC) converting it to IP3 and DAG
50
wHAT DOES dag ACTIVATE
PROTEIN KINASE c
51
wHAT DOES ip3 TRIGGER
Release of Ca2+ - also required for protein kinase C activation
52
How can Ca2+ concentration increase
1. Influx of Ca2+ from outside cell via Ca2+ channel proteins in the plasma membrane
2. Release of Ca2+ from intracellular stores I.e., endoplasmic reticulum (ER), sarcoplasmic reticulum (SR) and mitochondria (caused mainly via IP3)
53
What controls Ca2+ concentration
ATPase pumps in:
1. The plasma membrane (pump out Ca2+)
2. ER, SR and mitochondrial membrane (sequester Ca2+ back into organelle)
54
Structure of calmodulin
Has 4 Ca2+ binding sites
55
What activates calmodulin
when [Ca2+] increases above 500nM
56
How can termination of signaling events occur
1. Eliminate extracellular signaling molecule – enzymatic degradation
2. Deactivate signal transduction proteins – dephosphorylation by phosphates
3. Remove activated receptor from cell membrane by endocytosis
4. Receptor and signal molecule (ligand) are internalized: either the receptor and signaling molecule are separated and the receptor is recycled to surface and ligand destroyed Or the receptor and ligand are both destroyed.
57
How does a signaling molecule exert its effects
Binds to its specific receptor. l can also influence response by: regulating the number of receptors, synthesizing different isoforms of the receptor.
58
What is an agonist
A molecule that binds and activates a receptor, including signaling and a biological response e.g., native ligands and drugs
Full agonist: full activation
Partial agonist: partial activation
59
What is an antagonist
A molecule that binds to a receptor, but does NOT induce signaling and a biological response e.g., native ligands and drugs
60
What are the 3 types of cell surface rceeptors
1. Ion channel-linked receptor (ionotropic receptors)
2. G protein coupled receptor (metabotropic receptors)
3. Enzyme-linked receptor – intrinsic enzyme activity. Recruit enzyme from cytoplasm
61
How do ion channel-linked receptors work
Act as gates. Ligand binding causes receptor to change shape and open gate. Allows ion flow passively through channel
62
How can ion channel linked receptors be modified
channel blockers (physically block channel) or channel modulators (bind to channel and enhance or inhibit opening)
63
Structure of GPCR
1. Extracellular ligand binding region
2. Seven alpha helices that span the membrane
3. Intracellular portion interacts with a trimeric G protein
64
What are GPCRs
Bund an enormous range of extracellular signaling molecules. Mediate a wide array of physiological processes (including odorant detection)
65
What do GPCR utilise
trimeric G proteins to relay the signal
66
What are the 3 subunits of trimeric G proteins
alpha, beta and gamma
67
What does the G alpha subunit bind to
GDP/GTP and has the GTPase activity
68
Signal relay via the GPCR
1. Binding if ligand alters the conformation of the receptor
2. G alpha units binds to receptor
3. Binding of G alpha protein allows release of GDP and its exchange for GTP
4. Alpha subunit is active and dissociates from the beta and gamma units
5. Both active G alpha subunit and beta gamma complex can now interact with effector molecules to relay the signal – focus on G alpha subunit
69
How does the G protein switch off
1. G alpha subunit hydrolyses the GTP to GDP – occurs in seconds – can use RGS protein to aid hydrolysis
2. G alpha dissociates from effector molecule
3. Alpha subunit having returned to its original GDP inactive conformation can reassemble with the beta gamma complex to form inactive trimeric G protein
70
G alpha s protein (class of g protein) effector and effect
adenylyl cyclase - stimulation -> increase in cAMP
71
G alpha j protein (class of g protein) effector and effect
adenylyl cyclase - inhibition -> decrease in cAMP
72
G alpha q protein (class of g protein) effector and effect
phospholipase c - stimulation - > increase in DAG and IP3
73
What determines the G alpha class
by which effector molecule the G alpha subunit couples with and the resulting effect
74
Example of dysregulated G protein signaling
1. Cholera toxin binds to G alpha s and fixes it in GTP bound conformation
2. Over stimulation of adenyl cyclase and cAMP production
3. Downstream signaling effects transporters involved in ion transport leading to water loss
75
Example - oxytocin using G alpha q class
activate phospholipase C to induce Ca2+ mediated events to influence behavioral changes in the brain. LTP = long term potential
76
What do receptor tyrosine kinases consist of
Extracellular domain which binds the ligand (mainly growth factors). Transmembrane domain. Intracellular or cytoplasmic domain which contains the tyrosine kinase site - a tyrosine kinase adds phosphate groups from ATP to only tyrosine residues on target proteins
77
Activation of receptor tyrosine kinases
1. Requires dimerization of two receptor monomers
2. Activates the tyrosine kinase in each receptor
3. Kinase phosphorylates tyrosine on opposite receptor tail I.e., transphosphorylation
4. Recruitment/binding of adaptor and/or effector signaling molecules directly to the phosphorylates tyrosine's to initiate signaling
78
Example of key adaptors of RTKs
Grb2, Shc, IRS-1
79
Example of key effectors of RTKs
P13-kinase, phospholipase C
80
What do RTKs commonly utilise
the monomeric G protein Ras to relay the signal. Activated receptor either directly or indirectly (via an adaptor protein) binds and activates the GEF for Ras, thereby activating this key signaling molecule
81
Regulation of glucose uptake via activation of the insulin receptor
1. Glucose transporters I.e., GLUT-4 are stored in walls of cytoplasmic vesicles
2. Insulin induced IRS-1/PI-3 kinase/PKB signaling triggers vesicle translocation to the plasma membrane
3. Vesicle fuse with membrane where they take up glucose and pass it into the cell
82
What do cytokines like and what is the solution
Cytokine receptors lack intrinsic kinase activity so they recruit soluble tyrosine kinase I.e., JAK .
83
Janus kinase mechanism of activation
Ligand binding e.g., prolactin causes:
1. Receptor dimerization and JAK recruitment and activation
2. JAKs phosphorylate each other and the receptor
3. Recruitment of STAT transcription factor to phosphorylated tyrosine residues on the receptor
84
Nuclear receptors
lipid soluble molecules such as steroid hormones or molecules thta can pass through the ilayer bind to them. Exert its effects by affecting gene transcription
85
What does a nuclear receptor contain
1. A ligand binding domain
2. A DNA binding region - bind to ‘response elements’ in the promoter region of target genes
3. N terminal variable region which can be modified by other molecules to enhance transcriptional abilities
86
Where is cortisol produced
in the adrenal glands in response to stress
87
How does cortsiol mediate gene transcription
Passes through lipid bilayer and binds to its cytoplasmic nuclear receptor
*Ligand-bound receptor translocates to nucleus
*Binds to regulatory response elements in target gene to drive gene transcription
88
Plant signaling distances
1. Long distance (endocrine) - Slow via vascular system i.e. xylem and phloem
2. Short distance (paracrine) - Most common 3. No distance! – same cell (autocrine
89
Transport into plant vascular systems
Via active transport via transport proteins. Passive - freely diffusible. Via plasmodesmata
90
Juxtacrine signaling via plasmodesmata
Comprised of cytoplasmic channels linking adjacent cells
30-60 nm in diameter (cf. gap junctions with 1.5nm diameter) - Allows passage of both small molecules and macromolecules
Aids in electrical signalling between plant cells
91
What does electrical signaling in plants allow for
rapid long distance communication
92
Electrical signaling in venus fly trap
Stimulation of sensory trigger hairs activates mechano-sensitive ion channels. Lead to depolarization of membrane and generation of an action potential. Changes turgor pressure in hinge cells, causing closure of leaf lobes
93
How is signal transduction in plants similar to animals
1. Membrane enzyme-linked receptors and intracellular receptors, with and without kinase activity (but negligible existence of GPCRs and G proteins)
2. Use mainly serine/threonine kinases *
3. Intracellular signalling molecules e.g. lipid signalling molecules, Ca2+
94
Ethylene
Functions include fruit ripening and leaf abscission. Can pass through cell walls or diffuse through air
95
Where are ethylene receptors found
in the membrane of the endoplasmic reticulum and Golgi
96
What happens in the absence of ethylene
the ethylene receptor is activating a kinase –promoting the destruction of the transcription regulator
97
What does deactivation of ethylene receptor do
allows the transcription of ethylene sensitive genes
98
What can plants detect
the direction, intensity and wavelength (colour) of light
99
What are the 2 major classes of photoreceptor in plants
1. Blue-light receptors (3 types): Contain either cryptochromes, phototropin or zeaxanthin as photopigments. Cell surface receptor
2. Phytochromes (red light): Intracellular receptor
100
What does each phytochrome contain
Exist as two subunits and each has: A light detecting pigment or chromophore and a region that has kinase activity
101
How do phytochromes modulate gene expression
1. Translocating to the nucleus
2. Either directly binding to and activating a transcription factor
3. indirectly by phosphorylating transcription factors
102
What is apoptosis
A process seen in multicellular organisms by which specific cells are killed and removed for the benefit of the organism. 60 billion adult human cells per day die via apoptosis.
103
Why is apoptosis essential for animal development
removal of redundant structures and embryogenesis (e.g., sculpting of limbs)
104
How doe apoptosis maintain homeostasis in organisms
regulation of cell numbers, degenerative diseases (e.g., neurodegenerative disorders, ischemic heart disease or autoimmune diseases), diseases of over-proliferation (e.g., solid tumors, leukemia)
105
Apoptosis eliminates specific cells that are damaged beyond repair due to:
1. DNA damage – when repair mechanisms cannot cope with damage
2. Accumulation of misfolded proteins – causes endoplasmic reticulum stress and cell death. Linked with neurodegenerative disorders
3. Cells infected by certain viral agents – limits spread of infection
106
Morphological features of apoptosis
1. Ultrastructure changes (note: these changes are irreversible once apoptosis is triggered)
2. Cell shrinkage
3. Chromatin condensation
4. Fragmentation of intracellular contents and membrane blebbing
5. Formation of apoptotic bodies (ABs) - membrane – bound portions of cytoplasm and organelles
6. Phagocytic ingestion of Abs and degradation
107
What mediates apoptosis
a family of proteases called capases
108
Features of capases
cysteine at active site, cleaves target proteins at specific aspartic acids, synthesized as inactive procapases
109
What activates capases
Activated by proteolytic cleavage at own aspartic residues
110
What do initiate capase do
undergo autocleavage, activates other capases
111
What do effector capase do
Activates other effector capases after cleavage by initiator capase. Cleavage cellular proteins.
112
What nuclear effects are seen during apoptosis
hallmark cleavage of chromosomal DNA. Cleaves a protein that blocks endonuclease action so DNA can be cute into internucleosomal units of 180-200 base pairs
113
Apoptic cells on electropheresis
show DNA 'laddering'
114
What is hosphotidylserine
the key 'engulf me' signal
115
which cells recognise the 'engulf me' signal
phagocytes (macrophages and neutrophils) on cell surface of apoptotic bodies
116
Where is phosphotidylserine usually found
in inner leaf of plasma membrane - in apoptosis some molecules move to outer leafelet
117
What mediates phosphotidylserine flipping
Action of capases activate scramblase (Xkr8) which mediates PS flipping
118
What prevents cells from dying
trophic factors (e.g., induced signaling I.e., growth factor / survival factor withdrawal )
119
What does the activation of the intrinsic pathway depend on
upon the release of cytochrome c from the mitochondria - regulated by a balance between molecules that promote apoptosis and those which inhibit apoptosis
120
Pro-apoptotic molecules
BAX, BAK, BAD
121
Anti-apoptotic molecules
BCL-2, BCL-XL
122
How does BCL-2 inhibit apoptosis
by preventing release of cytochrome c from the mitochondria by blocking action of BAX and BAK
123
How does BAX/BAK promote apoptosis
by forming channels in the outer mitochondrial membrane to allow cytochrome c release
124
Events involved in the mitochondrial induction of apoptosis
1. apoptotic stimulus
2. Release of cytochrome C which activates adaptor protein
3. assembly
4. recruitment of procapase 9 molecules
5. activation of protocapase 0 within apoptosome
125
Survival factor or growth factor signaling suppresses apoptosis by ...
Increasing the transcription and translation of anti-apoptotic molecules
Signal transduction kinases (e.g., protein kinase B) which are activated by stimulation of trophic receptors, phosphorylate and inactivate pro-apoptotic molecules
126
Why is the extrinsic apoptosis pathway used
Used by cells of the immune system to kill their targets e.g., cancer cells. Pathogen-infected cells
127
What initiates the extrinsic pathway
Initiated by death ligands on/or secreted by the immune cells, binding to their receptors on the target cell
128
What does the T-lymphocyte have
death ligands FasL on surface which interacts with Fas (death receptor) - trimerized receptors use the adaptor FAAD to mediate autoactivation of initiator procaspase 8
The Dynamic Cell
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