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Cell Biology > Module 6 - Cell Signalling > Flashcards

Module 6 - Cell Signalling Flashcards

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1
Q

Dictyostelium Cycle between uni- and multicellular

A

Dictyostelium dicoideum (slime mold)

  • eukaryote
  • transitions from collection of unicellular amoebae into multicellular slug then into fruiting body
  • multicellular slug migrates towards heat, light and humitidy to find food
  • in suitable environment, anterior end forms stalk and posterior end forms spores of fruiting body
  • feed on bacteria like E.coli
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2
Q

signals for unicellular aggregation

A
  • food is abundant, single-cell amoebae divide via mitosis

- when food runs out, starvation initiates series of events leading to aggregation of the amoebae

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3
Q

Aggregation of amoebae

A
  • Occurs in response to production of AMP by starved cells, form a migrating multicellular collection called a slug.
  • Once the slug finds suitable nutrient rich environment, it stops and differentiate
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4
Q

fruiting body

A

-Formed from posterior cells, contains spores with a hard cell wall allowing spore to remain dormant for long periods of time

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5
Q

food availability of amoebae

A

when food is available, spores will germinate to form new single celled amoebae

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6
Q

signal for amoebae aggregation

A

cAMP

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7
Q

cAMP receptor

A

a transmembrane protein called G-protein coupled receptor or GPCR
extracellular domain of receptor binds to cAMP activating the receptor

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8
Q

response of cAMP binding to receptor

A

cells reorganize the intracellular actin network to move towards source of the signal. When cAMP signal moves, the cell responds by changing its direction of movement

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9
Q

how amoebae moves

A

dynamic filopodia extend outward to allow movement.
- signalling initiates actin reorganization including nucleation, polymerization, and depolymerization to enable movement

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10
Q

Mutation in the clathrin heavy chain in dictyostelium

A
  • means that the cells are unable to form the vesicles necessary for transport of proteins to the cell membrane
  • no net movement of the cell towards the signal
  • in the absence of protein transport, the GPCR is not transported to the cell surface and there is no receptor for cAMP and the cell is unable to respond to the signal
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11
Q

Human Neutrophils movement towards chemical signals

A

able to respond to a signal produced by bacteria that have invaded our bodies
a receptor on the surface of the neutrophil binds to this chemical signal, activating a series of internal changes that facilitate directional movement
eventually the neutrophil is able to capture and engulf the bacterium in a process of endocytosis

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12
Q

neutrophil cell receptor and molecule

A

signal produced by the bacteria is a protein containing the tripeptide formylated methionine, leucine and phenylalanine.
- neutrophil has a cell surface GPCR that specifically recognizes the fMLP peptide (fMLP receptor)

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13
Q

cell-cell signalling definition

A

transmitting information from one cell to another and inducing a change in behaviour or response
signal is only useful if there is a response to the signal
must include production and release of a signal, the perception of the signal, interpretation of that signal inside the cell and a resulting change in behaviour

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14
Q

principles of signal transduction

A

binding of the signal activates the receptor which initiates a cascade of chemical events inside the target cell that interpret and transduce the signal

  • this culminates in some changes in target cell behaviour
  • responses include: changes in transcription, cell movement or growth, cell differentiation, and changes in metabolism corresponding to enzyme activation and inactivation within the cell
  • signal must be removed to terminate the target cell response
  • many cells might be exposed to a signal, but only the target cells with the appropriate receptor will be able to respond
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15
Q

receptor signal interactions

A
  • receptor signal binding follows same principles of molecular complementarity as any protein ligans interactions
  • there is a specific and high affinity interaction between the receptor and the signal that is determined by molecular complementarity between the faces of the molecules
  • complementary shapes allow the interacting surfaces of the 2 molecules to come close together
  • collection of non-covalent interactions provide specificity and high affinity
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16
Q

essential aa residues in receptor signal interactions

A
  • are aa that are necessary for the receptor signal binding

- a signle aa change at any of these residues can reduce or eliminate signal binding and therefore disrupts signalling

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17
Q

result of receptor signal interactions

A

there is a conformational change in the intracellular domain of the receptor which includes the signal transduction pathway and ultimately the cellular response

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18
Q

2 levels that specificity of the signal response is achieved at

A

1) the specificity of the ligand for binding to the receptor
2) the specificity of the intracellular response that is mediated by the signal transduction pathway

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19
Q

cell specificity of the intracellular responses

A
  • 2 different cells may respond to the same signal by activating different TFs
  • some cells may respond to the same signal by either moving or altering the metabolic activity
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20
Q

signal transduction pathway

A
  • the collection of intracellular steps required to translate an extracellular signal into a cellular response
  • the specificity of the response will be determined by the internal STP
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21
Q

different responses for STP

A

gene transcription, cell division, growth, differentiation, changes in shape, movement, changes in metabolism

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22
Q

Fast responses

A

changes in enzyme activation

  • extracellular signal binds to a membrane associated receptor
  • a cytosolic enzyme is activated in response to activation of receptor through modifications like methylation, acetylation or phosphorylation
  • fast response because the cell is able to quickly respond to the signal by simply changing the activity of a cellular protein that is already present in the cell
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23
Q

slow responses

A

changes in gene transcription

  • change in protein levels
  • soluble receptor is in the cytosol and the signal is able to pass through the cell membrane
  • activation of the receptor leads to receptor transportation into the nucleus, where it acts directly or indirectly as a transcriptional activator producing mRNAs
  • mRNAs translated to increase protein levels
  • slow response because the response depends upon transcirption, translation, protein folding, protein modifications and each step takes time before seeing a change in cellular response
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24
Q

2 ways to assay a signal

A

affinity of the receptor for a signal can be measured in the same way that protein ligand affinity is measured

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25
Q

Kd

A

dissociation constant: [L] required to have half maximal binding

  • represents receptor signal affinity
  • can be compared to concentration of signal required to achieve a physiological response
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26
Q

maximal / half maximal physiological response

A

can be measured (all cells respond) and a 1/2 maximum response can be calculated
the [L] to achieve half of the maximal physiological response is much lower than the [L] required to fill 1/2 of the receptors
this suggests that the signal is amplified inside the cell and implies that very little signal is required to exert a response

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27
Q

endocrine signalling

A

secreted signals are released into the circulatory system
-in this way, cells throughout the body are exposed to the signal
signalling molecule = hormone, target = distal
only cells that have the appropriate receptor can respond to the signal
many different cells in different tissues can respond to the same signal at the same time

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28
Q

paracrine signalling

A

secreted cells are released in the extracellular space. where they can diffuse to neighbouring cells
-signalling molecule = GF and NT

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29
Q

integral membrane protein signalling

A

proximal signalling where the signalling cell and target cell are in direct contact with one another

  • both the signal and receptor protein may be transmembrane proteins on different cells
  • interaction between the signal and receptor requires that the 2 cells are attached together
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30
Q

plasmodesmata in plants

A

communication by sharing cytosolic messengers
-junction between 2 neighbouring cells that span the cells membranes and the cell wall
effectively connect the cytoplasm of neighbouring cells, allowing messengers to move very quickly from one cell to the next
-these connections form a vascular system within plants in which signals produced in the root can be transported up to the leaves

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31
Q

gap junctions in animals

A
  • channels connecting the cytoplasm of neighbouring cells that allows. the fast diffusion of small molecules from one cell to another
  • in this way, one cell may respond to a primary extracellular signal by producing an internal secondary messenger which can then diffuse from one cell to another to exert the same response and coordinate the behaviour of a series of cells
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32
Q

autocrine signalling

A

process in which a cell communicates with itself

  • signalling cells and target cell are the same
  • signalling molecule = secreted (e.g. GF that are produced to induce cell division or stop cell division depending upon internal and external conditions
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33
Q

classes of cell-surface receptors

A 
G-protein coupled receptors (GPCR)
cytokine receptors
receptor tyrosine kinases (RTK)
TGF beta receptors
hedgehog (Hh) receptors
Wnt receptors
Notch receptors
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34
Q

cytokine receptor of the JAK/STAT pathway

A

controls production of RBC by phosphorylation of effector protein

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35
Q

receptor tyrosine kinase (RTK)

A

linked to phosphorylation cascade through a small G-protein, Ras to regulate gene expression

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36
Q

GPCR

A

activates an effector protein inside the cell to produce a secondary messenger cAMP that ultimately regulates cell metabolism

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37
Q

RBC production

A

2 million RBC produced / second in adults

  • cells develop at the bone marrow and circulate for about 4 months before digested and recycled by macrophages
  • RBC are replaced when progenitor cells stop dividing and differentiate
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38
Q

Erythropoietin (Epo)

A

signal for the maturation of the RBC

  • cytokine
  • expression of Epo is regulated by an oxygen binding TF in kidney cells
  • Epo is released into circulatory system
  • only erythrocyte progenitor that carry a receptor (EpoR)
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39
Q

EpoR

A

cytokine receptor that is linked to JAK/STAT signal transduction pathway
-response include inhibition of cell death, changes in patterns of gene expression and differentiation

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40
Q

components of Epo pathway

A

Signal = cytokine, erythropoietin (Epo)
receptor = Epo receptor (EpoR)
Intracellular signal transduction pathway = Jak kinases and STAT TFs
Cellular response = responding change in target cell behaviour, transcription of STAT target genes; inhibition of apoptosis
-EpoR is inactive as a monomeric single-pass transmembrane protein. The Epo signal interacts with 2 EpoR initiating dimerization
-cytosolic domains of 2 cytokine receptors associated indirectly through a single Epo molecule

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41
Q

EpoR dimerization and autophosphorylation

A

cytosolic, transmembrane and extracellular

  • each EpoR is associated with a JAK kinase on its cytosolic domain
  • a JAK kinase in the phosphorylated state is inactive, with very weak kinase activity
  • dimerization of the receptors by Epo brings the 2 JAK kinases close together
  • weak kinase activity is enough to phosphorylate a neighbouring JAK kinase, called autophosphorylation
  • phosphorylation of the activation lip on JAK kinase activates kinase activity
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42
Q

JAK kinase

A
  • JAK kinases have many targets of phosphorylation including tyrosine residues on the intracellular domain of the receptor
  • JAK kinase is specifically a tyrosine kinase meaning that only tyrosine residues are phosphorylated
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43
Q

phosphorylation of protein docking sites

A
  • activation of the receptor initiates a cascade of intracellular events
  • phosphorylated docking sites are available for protein-protein interactions, including binding of the STAT TFs
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44
Q

STAT interaction domain and phosphorylation

A
  • STAT has a protein-protein interaction domain called SH2 that specifically recognizes phosphorylated tyrosine residues
  • STAT monomers are inactive
  • dimerization and activation. depend upon phosphorylation
  • by accumulating on the receptor docking sites, the STAT protein is proximal to the JAK kinase
  • now STAT is a target of phosphorylation of JAK kinase
  • phosphorylation allows dimerization and changes the conformation such that a nuclear localization sequence is unmasked
  • now STAT dimer can be transported into the. nucleus
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45
Q

SH2 protein binding domain

A
  • a protein-protein interaction domain
  • is essential to the function of the cytokine signalling lathway
  • the domain has no enzymatic function, simply allows a protein to bind to specific target substrates
  • may have the effect of relocalization of a protein within the cytoplasm, as we saw with STAT, or linking together proteins in a pathway
  • SH2 domain did not change but its targets did: either the docking site tyrosine was phosphorylated or unphosphorylated
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46
Q

SH2 affinity

A

-SH2 binds with high affinity and specificity to this sequence when the tyrosine is phosphorylated but with low affinity when tyrosine is unphosphorylated

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47
Q

protein-protein interaction domains

A
  • many examples, all responsible for linking together 2 proteins
  • in some cases, binding is dependent upon reversible modifications to the target peptide
  • SH2, PTB and 14-3-3 domains bind peptides containing phosphorylated tyrosine with high affinity, but not the corresponding phosphorylated peptide
  • allows protein-protein binding to be reversible
  • other domains bind to sequences that are not modified ex. PDZ domains bind hydrophobic residues at the C-terminus and SH3 and WW domains bind proline-rich domains (not reversible)
48
Q

erythrogenesis

A

associated with activation of the STAT5 FT that regulates many genes necessary for the differentiation of mature RBC
-while bone marrow is primary source of erythrogenesis, RBC are also formed in the liver, evident during development where all RBC are formed in the fetal liver

49
Q

target genes of STAT TFs

A

one example: gene Bcl-XL which codes for the Bcl-XL protein that is an inhibitor of apoptosis
-by inhibiting apoptosis, the erythroid progenitor cells persist and differentiate

50
Q

mutations in EpoR

A

wild type, can see bright red abdomen because fetal liver creating RBC
mutation in pathway, mouse is homozygous for loss of function allele of the EpoR gene, liver is still in tact, but no RBC are being made
-mutations for genes coding for proteins required at different steps in the cytokine pathway can cause same phenotype, including mutations in the genes coding for JAK kinase, the Epo signal, the STAT protein or the Bcl-XL protein

51
Q

lethal effects of erythrogenesis

A

disabling erythrogenesis by eliminating the receptor is lethal

  • failing to turn off the signal can also be. lethal
  • continual activation of the cytokine pathway leads to overproduction of RBC
52
Q

Effects of elevated hematocrit (or RBC count)

A

increase the [ ] of RBC in the blood, increases the viscosity of the blood

  • result could be blockage of narrow capillaries of the circulatory system
  • blockage can result in a stroke or heart attack
53
Q

use of Epo with athletes

A

some athletes intentionally increase their hematocrit by taking exogenous erythropoietin (aka Epo doping) the increase RBC count increases the capacity to carry oxygen
-this is a dangerous practice that can be lethal

54
Q

short term mechanisms that turn off the JAK/STAT pathway

A
  • reversing phosphorylation: a phosphatase dephosphorylates modified aa residues, the SHP1 phosphatase has 2 SH2 domains. that allow it to doc at the same TF, phosphatase activity is stimulated by binding of the SH2 domains to its phosphorylated ligands, localization of SHP1 to the docking sites mediates dephosphorylation of JAK kinase, turning off the signalling pathway
  • this is short term inactivation because removal of SHP1 allows fast reactivation of JAK kinase
55
Q

long term regulation of erythrogenesis

A

1) through SOCS protein; SOCS is able to bind to the phosphorylated docking sites via SH2 domain, it is expressed in response to high oxygen levels in the body
- multiple SOCS bind to the docking sites blocking access to the sites by STAT
- SOCS is an E3 ubiquitin ligase that targets JAK kinase for ubiquitinylation and degradation through the proteosome
- removal of JAK kinase turns off the signalling pathway
- reactivation is slow as it requires the expression of new JAK kinase proteins
2) receptor recycling and signal release; many signalling pathways can be turned off when the receptor is internalized through endocytosis
- receptor can be recycled back to the surface of the cell, but if Epo levels have gone down, the receptor will not be reactivated

56
Q

mutations in EpoR gene

A

create truncated versions of the EpoR

  • proteins have shorter docking sites
  • this has been associated with a decrease sensitivity to the negative regulatory of the JAK-STAT pathway SHP1 and SOCS
  • result is a higher hematocrit
  • individuals carrying this mutation show RBC levels comparable to a person who is taking exogenous Epo, but they are not Epo doping
57
Q

Receptor tyrosine kinase (RTK) and Ras

A

signals: nerve growth factor (NGF), platelet derived growth factor (PDGF), epidermal growth factor (EGF), insulin
cell responses: cell differentiation, division, survival/apoptosis, metabolism
-in all cases, the hormone will interact with a transmembrane receptor tyrosine kinase, and activate the intrinsic kinase activity of that receptor

58
Q

components of RTK and Ras pathway

A

RTKs have an extracellular signal binding domain, a single pass transmembrane domain and intrinsic kinase activity on the cytoplasmic domain of the protien

  • it is ligand binding that leads to dimerization of the receptor and autophosphorylation of the kinase domain
  • intracellular signal transduction pathway is much longer than in the cytokine pathway and involves activation of an intracellular, membrane-anchored protein called Ras
  • regulation of Ras requires proteins that link it to the activated RTK including adaptor proteins (GRB2) and the Ras effectors GEF and GAP
  • Ras G-protein activation leads to a kinase cascade that accumulates inactivation of the MAP kinase
  • MAP kinase modulates cell behaviour by phosphorylation TFs and changing patterns of gene expression
59
Q

RTK signal binding and receptor dimerization

A

ligang binding leads to dimerization of the transmembrane receptors
-EFG binds to each of the receptors, changing the conformation of the extracellular domain and inducing dimerization

60
Q

RTK phosphorylation of docking sites

A

monomeric RTK receptor has poor intrinsic kinase activity

  • dimerization has the effect of bringing 2 kinase domains very close together so that even the weak kinase activity can lead to phosphorylation of neighbouring activation lip
  • phosphorylation of the activation lip increases kinase activity allowing phosphorylation of more target proteins
  • tyrosine residues on intracellular docking sites of the receptor are targets of kinase activity
  • phosphorylated docking sites are potential binding sites for protein-protein interaction domains such as SH2 and PTB
61
Q

adaptor proteins binding P-tyr at docking sites

A

adaptor proteins carry 2 or more protein interaction domains that allow the protein to act as linkers between other proteins
-this has the effect of indirectly linking proteins to the receptor

62
Q

scaffold proteins

A

also, adaptor proteins that have multiple protein-protein interaction domains and assemble proteins in an ordered scaffold

63
Q

GRB2

A

an adaptor protein with 3 protein-protein interaction domains

  • 1 SH2 domain and 2 SH3 domains
  • SH2 domain recognizes p-tyr on the RTK docking site,
  • SH3 domains recognize pro-rich sequences, binds SOS
  • binding of SH2 domains is dependent upon the reversible phosphorylation of the docking sites
64
Q

SH3 protein interaction domain

A

-highly specific interactions between SH3 domains and proline-rich target peptide, 2 shapes complement one another

65
Q

GTP bound G-protein in the active conformation

A
  • arms or switches interact specifically with the terminal phosphate on the GTP in the nucleotide binding site
  • GTP fits specifically within the binding pocket of the G-protein, and the negative charge on that terminal phosphate interacts with glycine and threonine residues on each switch, pulling the switches together
  • the intrinsic enzymatic activity of the G-protein is distinct from the ON and OFF state of the protein
  • it is not the GTPase activity that is turned ON and OFF but this GTPase activity is required for turning the protein “OFF”
  • receptor activation leads to activation of G-protein Ras
66
Q

G-protein or GTPase switch protein regulation

A

these are all GTP binding proteins

  • when bound to GTP, the protein is active, but when bound to GDP, the protein is inactive
  • the G-protein also has intrinsic GTPase activity
  • GTPase activity is always active, through its activity can be modulated by other factors
67
Q

GDP-bound “OFF” state

A
  • GTPase hydrolysizes the terminal phosphate of GTP, releasing inorganic phosphate (Pi)
  • GDP now remains in the nucleotide binding pocket
  • in the absence of that terminal phosphate, the switches are no longer held inward and instead fold out
  • g-protein is off
  • GDP has a low affinity for the nucleotide binding pocket so GDP will leave
  • g-protein will remain in its off state until a GTP comes in the binding pocket and activates the protein again
68
Q

GEF

A
  • guanine nucleotide exchange factor
  • activation accelerated by GEF
  • promotes dissociation of GDP and allows GTP to enter the nucleotide binding pocket
  • in this way, GEF acts as an activator of G-protein activity
69
Q

GAP

A
  • GTPase activating protein
  • deactivated by GAP
  • accelerates intrinsic GTPase 100-fold (or more) and rapidly inactivates the G-protein (in association with Ras)
  • promotes inactivation of the G-protein by enhancing the intrinsic GTPase activity
70
Q

GDI

A
  • inactivated by GDI
  • guanine nucleotide dissociation inhibitor
  • increasing the affinity of the nucleotide-binding pocket for GDP, keeping the G-protein inactive or “OFF”
71
Q

Ras-GDP/GTP cycle

A

the “OFF” state is maintained when GDI promotes GDP binding, counteracting this is a GEF protein which promotes the dissociation of GDP

  • when GDP leaves the binding pocket, GTP readily comes in
  • GTP binding happens quickly because there is a high [ ] of GTP in the cell and the nucleotide. binding pocket has a high affinity for GTP
  • once the protein is bound to GTP, it is “ON”
  • GAP promotes the inherent GTPase activity of the G-protein which hydrolyzed GTP to GDP
  • now the G-protein is “OFF” and can no longer interact with the target protein
  • G-protein remains on for a fixed amount of time depending on the presence of the modulator protein GAP
  • the length of the time that the G-protein is on determines how much of the target protein is activated and for how long it is activated
  • elimination of GAP would result in increased duration of the active G-protein, signalling would persist for a long time
72
Q

SOS GEF

A

GEF that interacts with the Ras G-protein

  • SOS protein was the protein that was relocated to the cell membrane through indirect association with the activated RTK receptor
  • SH3 domains of the GRB2 adapter protein hold SOS close to the membrane
  • this has the effect of bringing SOS proximal to the membrane-anchored Ras G-protein
  • the interaction of SOS with Ras promotes the release of GDP and the subsequent binding of GTP, thus activating Ras
73
Q

conformation states for Ras protein

A

1) inactive Ras-GDP
2) SOS binding that displaces GDP
3) active Ras-GTP

74
Q

GAP for Ras: NF1 (neurofibromatosis)

A
  • GAP protein NF1 enhances the intrinsic GTPase activity of Ras and accelerates the rate of hydrolysis of GTP thus inactivating Ras
  • presence of NF1 will shorten the length of time the G-protein is active
  • a loss of function mutation in the NF1 gene eliminates GAP protein in this pathway and increases the length of time that Ras is active, allowing signalling to persist longer than it should
  • loss of function mutations are associated with tumorigenesis in neural systems
  • in pathways regulating mitosis, this can mean increased rates of cell division
75
Q

position of Ras in the pathway

A

Ras activation is downstream of receptor activation meaning that Ras activation occurs after RTK activation and that Ras activation is dependent upon RTK activation

76
Q

Adding EGF (control)

A
  • signal binds to the EGF receptor on the surface of the cell and induces cel division
  • there is a corresponding increase in cells in culture
77
Q

adding antibody to Ras

A
  • removes ras function
  • adding antibody blocks the ability of Ras to interact with its target substrate and eliminates Ras activity
  • when EGF is added there is no cell division
  • blocking a downstream step (Ras) eliminates signalling
78
Q

replace Ras with constituitively active form of Ras (Ras-D)

A

-protein variant lacks GTPase activity and so the G-protein is always active
-no EGF is added and the RTK is not activated
the cells are still rapidly dividing
-suggests that Ras is downstream of the RTK because we have bypassed the effect of an inactive RTK by having a constitutively active downstream component
-a constitutive dominant mutation in Ras is associated with the formation of tumours in many different cell types

79
Q

failure in GTPase activity in Ras

A

this mutation leads to the elimination of a single glycine residue in Ras that blocks binding of the GTPase accelerating protein

80
Q

Failure in GAP protein

A

NF1 is another component that leads to uncontrolled cell division when absent

81
Q

Her2

A

an RTK that has been linked to hereditary forms of breast cancer

  • wild type Her2 is a receptor for the EGF signal
  • mutant variant for Her2 does not respond to the signal and instead is always activated because the mutant Her2 is always dimerized, even in the absence of that signal ligand
  • constitutive dimerization of Her2 receptor leads to uncontrolled cell division
82
Q

Ras activation leading to Raf activation

A

phosphorylated residues on Raf are bound by the 14-3-3 adapter protein

  • this holds Raf in an inhibited conformation
  • binding of Raf to Ras releases 14-3-3 binding and activates Raf
83
Q

Raf

A
  • Raf is a serine/threonine kinase protein that is at the top of a kinase cascade
  • aka MAP kinase kinase kinase
  • leads to phosphorylation of its target protein MEK
84
Q

MEK

A
  • aka MAP kinase kinase

- phosphorylates MAP kinase at 2 residues, tyrosine and threonine, activating this protein at the end of the cascade

85
Q

MAPK

A
  • MAP kinase is a serine/threonine kinase that dimerizes upon activation and is translocated to the nucleus where target transcription factors are phosphorylated and activated
  • a scaffolding protein acts to hold the proteins in this cascade close together in an ordered series
86
Q

Activation lip

A

contains threonine and tyrosine residues that are targets of the dual specificity MEK kinase

  • the change in conformation of the activation lip reveals the ATP and substrate binding pockets
  • this is similar to the mechanism we have seen in various kinase proteins
  • MAP kinase is a downstream target of all Ras-linked RTKs
87
Q

activation of transcription

A
  • targets of active MAP kinase dimer
  • P90 RSK kinase is phosphorylated in the cytoplasm, allowing it to be translocated into the nucleus
  • MAP kinase is itself translocated into the ncuels
  • once inside, the nucleus these 2 kinases phosphorylate a target TF
  • one is the tertiary complex factor (TCF) that is directly phosphorylated by MAP kinase, the other is the serum response factor (SRF) that is directly phosphorylated by P90 RSK
  • together these TFs bind to DNA sequence called the serum response element (SRE) which is an enhancer sequence upstream of a collection of genes
  • when the 2 TFs bind to the SRE and form this complex, they promote. the assembly of RNA polymerase and transcription of the target gene
  • many target genes contain the upstream SRE including c-fos
88
Q

c-fos

A

a gene that codes for another TF that enhances rates of transcription of genes required for regulating and turning on the cell cycle

89
Q

cellular responses

A

the change in cell behaviour at the end of the signalling pathway is transcriptional activation and the induction of cell division, differentiation and other cell behaviours

90
Q

amplification of the signal

A
  • the size of the protein shapes in each step of the pathway illustrates an important concept
  • each kinase enzyme can target multiple proteins
  • this allows amplification of the signal at each step
  • one EGF signal could lead to millions of activated ERK 1/2 MAP kinase proteins and in turn millions of copies of the target protein required for cell division
  • in this way, the signalling pathway is very sensitive to low concentrations of hormones that are secreted into the circulatory system and present at nanomolar concentrations
91
Q

GPCR structure

A

all GPCR share a common structure; 7 membrane-spanning domain which consists of 7 transmembrane alpha-helices that loop through the membrane of the cell

  • creates 4 extracellular segments which fold in the extracellular space to form the signal binding domain
  • creates 4 cytoplasmic segments which fold to form an internal domain that interacts with a trimeric G-protein
92
Q

Adrenergic stress response

A

Signal: catecholamines - epinephrine and norepinephrine
receptor: GPCR
intracellular transduction: effector enzyme = adenylyl cyclase, second messenger = cAMP
cellular response: the release of stored energy
-fast = enzyme activation
slow = activation of transcription

93
Q

catecholamines

A
  • water soluble signals circulating in the blood
  • most abundant are epinephrine (adrenaline) and norepinephrine (noradrenaline) and dopamine
  • release of the hormones epinephrine and norepinephrine from the adrenal medulla of the adrenal gland is part of the fight or flight response
  • binds 2 types of GPCR (B and a2) but induce different responses depending on which receptor it is bound to
  • breakdown of stored energy; glycogen in the liver = glycolysis, fatty acids in adipose tissue = lipolysis
94
Q

GPCR catecholamine receptor

A

-intracellular signalling pathway involves activation of the receptor associated trimeric g-protein and activation of an effector protein called AC that modulates the cytosolic concentration of cAMP

95
Q

beta adrenergic receptors

A

are stimulatory, allows coordinated respond to occur in different tissues in response to the same signal

  • liver and adipose tissue: glycolysis and lipolysis
  • heart muscle: increases contraction resulting in increase blood supply to tissues throughout the body
  • smooth muscle cells of the intestine: increase muscle cell relaxation so as not to expend unnecessary energy during times of stress, save energy for major locomotory muscles
96
Q

alpha2 adrenergic receptors

A

are inhibitory
-found in blood vessels in the skin, of smooth muscles, intestines, kidney: cause arteries to constrict, the blood supply is reduced to periphery

97
Q

energy during stress response

A

-the stress response increases the supply of ATP to cells through the breakdown of energy stores; glycogen and triglycerides

98
Q

membrane associated proteins

A

GPCR is in the inactive state during which it is not associated with the lipid anchored trimeric g-protein

  • trimeric g-protein follows the same principles of other monomeric g-proteins that we have seen: there is an active and an inactive state that is dependent upon guanine nucleotide binding
  • when the G-protein is bound to GTP it is active, when it is bound to GDP it is inactive
99
Q

activation of receptors

A

receprtor is activated through binding of the extracellular signal which induces a conformational change in the intracellular domain that allows it to interact specifically and with high affinity to the trimeric g-protein

  • interaction induces a change in conformation that causes the dissociation of GDP and allows binding of GTP in the nucleotide binding pocket
  • GTP bound G-protein is active
100
Q

activation of effector AC

A

trimeric g-protein dissociates, releaseing the G-alpha subunit

  • this activated subunit can move laterally in the cell membrane and interact with the effector enzyme
  • the activated effector will remain active for a short period of time, only while the G-protein is associated
  • the length of time of the activation is dependent upon the intrinsic GTPase activity of the G-protein
  • once GTP is hydrolyzed to GDP, the G-protein become inactive which in turn releases and inactivates the effector
101
Q

Beta adrenergic receptors mechanism

A

stimulatory receptors associated with stimulatory G-protein (Gs)

  • 3 subunits, a, B, y
  • Gs cycles b/w the active GTP bound. form and the inactive GDP bound form
  • upon receptor activation, Gsa-GTP dissociates from GBy then binds and activates adenylyl cyclase (effector enzyme and increase the intracellular [ ] of cAMP
  • intrinsic Gsa GTPase hydrolyzes GTP to GDP
  • Gsa-GDP reassociates with GBy, inactivating AC
  • hydrolysis of GTP to GDP inactivates Gsa and it dissocates from AC,
  • in the absence of an active AC, there are ubiquitous cytosolic enzymes that decrease the [ ] of cAMP
  • only by maintaining an active AC through receptor activation can the cytosolic [ ] of cAMP stay high
102
Q

alpha2 adrenergic receptor mechanism

A
  • same GB and Gy subunits as beta-adrenergic
  • different Ga, inhibitory Gia that inhibits AC
  • interacts with different region of AC catalytic domain
  • the a2 adrenergic receptor inhibit production of energy and energy usage
  • in the absence of AC there is no increase in cAMP
103
Q

activation and inhibition of AC

A

epinephrine can activate both receptors on different cell types in order to coordinate diverse responses as part of the fight or flight response

104
Q

Adenylyl cyclase enzyme

A

-converts ATP into cAMP with the release of diphosphate

as long as there is an active AC, the cell will maintain a high [ ] of cAMP

105
Q

degradation of cAMP by PDE

A

PDE (phosphodiesterase) is constitutively active in the cell and counteracts AC

  • PDE catalyzes the breakdown of cAMP into 5’AMP
  • even as active AC is making more cAMP, PDE is breaking down cAMP
  • as long as AC remains active, cytosolic cAMP remains high
  • as soon as AC is inhibited, there is a drop in cAMP [ ]
106
Q

secondary messengers

A

secondary messengers are small soluble molecules, intracellular signalling molecules

  • activation of signalling pathways leads to rapid but short lived increase in cytosolic [ ] of secondary messengers
  • the [ ] of secondary messengers fluctuates in the cell depending upon the activity of signalling pathways
  • in turn, it is the [ ] of the messenger that determines the activation or inactivation of the next step in the signalling pathway
107
Q

cAMP role as secondary messenger

A

responds to the GPCR signalling pathway

the [ ] of cAMP modulates the activity of target proteins including pKA

108
Q

pKA

A

cAMP -dependent protein kinases = protein kinase A

-pKA is a serine-threoinine kinase that phosphorylates a variety of target proteins

109
Q

activation of pKA

A

-tetrameric pKA is inactive
-2 regulatory subunits
-2 catalytic subunits
-regulatory subunits have nucleotide binding sites that bind cAMP
when cytosolic cAMP [ ] are low, there is no cAMP in these binding pockets which inactivates pKA due to the interaction of the pseudosubstrate domain of the regulatory subunit with the substrate domain of the catalytic subunit
-as the [ ] of cAMP increases, the binding sites on the regulatory subunits are filled
-this induces a conformational change in the pseudo-substrate domain of the regulatory subunit that releases the catalytic subunit

110
Q

regulation of pKA

A

R with cAMP: cAMP is bound and the pseudo-substrate retracts, allowing activation of the pKA enzyme (catalytic subunit)
R without cAMP: cAMP is released and the pseudo-substrate domain is extended and is able to block the substrate-binding domain of pKA, thus inactivating enzyme function

111
Q

how pKA regulation relates to stress response

A

response to the epinephrine signal is an increase in the supply of energy to many tissues in the body
-role of pKA is determined by the targets of pKA activity
in order to release ATP, body needs to supply glucose to the cells. The products of glycolysis (glucose metabolism) are pyruvate and NADH, used by the mitochondria to make ATP

112
Q

targets of pKA

A

glycogen: major storage form of glucose (polymer of glucose)
- synthesized by one set of enzymes (glycogen synthase)
- degraded by another set of enzymes (glyocgen phosphorylase)
- to release glucose from glycogen, need to inhibit glycogen synthase and promote glycogen phosphorylase

113
Q

net effect in muscle to epinephrine

A

glycogen is metabolized into glucose-6-phosphate (G-6-p)

  • this is the source of glucose for the cell
  • glycolysis produces pyruvate and NADH, substrates for ATP production in the mitochondria
  • the increase supplies of ATP can power the skeletal muscles and cardiac muscles in the fight or flight response
114
Q

net effect in liver to epinephrine

A

stored glycogen is metabolized to glucose-6-phosphate

  • this occurs by the indirect activation of glycogen phosphorylase, which catalyzes glycogen breakdown
  • pKA regulates this process by phosphorylation phosphorylase kinase that in turn activates glycogen phosphorylase
  • liver cells inhibit the production of more glycogen synthesis when pKA phosphorylates and inactivates glycogen synthase)
  • stimulation of glycogen breakdown through indirect activation of glycogen phosphorylase that degrades glycogen
  • G-6-P converted to free glucose, released to blood and transported to other tissues
  • this is a fast short term response to epinephrine that requires only the modification of enzymes already present in the cell
115
Q

activation of gene transcription

A
  • slow long-term response to this signalling pathway
  • pKA is in active state, catalytic subunit can be translocated into the nucleus where it can phosphorylate TFs including CREB which binds to the cAMP response element (CRE)
  • this is an enhancer sequence found upstream of many genes
  • when CRE is bound by CREB, it enables the assembly of the transcriptional machinery to initiate transcription
  • the target genes include those that are required for the production of glucose, including the genes for phosphorylase kinase and glycogen phosphorylase
116
Q

amplification of the signal

A

between the addition of the epinephrine signal and the production of glucose, there is a 10^8 fold amplification response
-the [ ] of epinephrine is about 1 in 10^10 molar in the blood system, but this is enough to activate a large scale response in cells at many locations across the body

Cell Biology (6 decks)

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