Module 6 - Cell Signalling

Question 1

Dictyostelium Cycle between uni- and multicellular

Answer 1

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

Question 2

signals for unicellular aggregation

Answer 2

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

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

Question 3

Aggregation of amoebae

Answer 3

• 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

Question 4

fruiting body

Answer 4

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

Question 5

food availability of amoebae

Answer 5

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

Question 6

signal for amoebae aggregation

Answer 6

cAMP

Question 7

cAMP receptor

Answer 7

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

Question 8

response of cAMP binding to receptor

Answer 8

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

Question 9

how amoebae moves

Answer 9

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

Question 10

Mutation in the clathrin heavy chain in dictyostelium

Answer 10

• 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

Question 11

Human Neutrophils movement towards chemical signals

Answer 11

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

Question 12

neutrophil cell receptor and molecule

Answer 12

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)

Question 13

cell-cell signalling definition

Answer 13

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

Question 14

principles of signal transduction

Answer 14

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

Question 15

receptor signal interactions

Answer 15

• 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

Question 16

essential aa residues in receptor signal interactions

Answer 16

• 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

Question 17

result of receptor signal interactions

Answer 17

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

Question 18

2 levels that specificity of the signal response is achieved at

Answer 18

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

Question 19

cell specificity of the intracellular responses

Answer 19

• 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

Question 20

signal transduction pathway

Answer 20

• 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

Question 21

different responses for STP

Answer 21

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

Question 22

Fast responses

Answer 22

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

Question 23

slow responses

Answer 23

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

Question 24

2 ways to assay a signal

Answer 24

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

Question 25

Kd

Answer 25

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

Question 26

maximal / half maximal physiological response

Answer 26

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

Question 27

endocrine signalling

Answer 27

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

Question 28

paracrine signalling

Answer 28

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

Question 29

integral membrane protein signalling

Answer 29

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

Question 30

plasmodesmata in plants

Answer 30

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

Question 31

gap junctions in animals

Answer 31

- 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

Question 32

autocrine signalling

Answer 32

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

Question 33

classes of cell-surface receptors

Answer 33

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

Question 34

cytokine receptor of the JAK/STAT pathway

Answer 34

controls production of RBC by phosphorylation of effector protein

Question 35

receptor tyrosine kinase (RTK)

Answer 35

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

Question 36

GPCR

Answer 36

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

Question 37

RBC production

Answer 37

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

Question 38

Erythropoietin (Epo)

Answer 38

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)

Question 39

EpoR

Answer 39

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

Question 40

components of Epo pathway

Answer 40

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

Question 41

EpoR dimerization and autophosphorylation

Answer 41

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

Question 42

JAK kinase

Answer 42

- 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

Question 43

phosphorylation of protein docking sites

Answer 43

- 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

Question 44

STAT interaction domain and phosphorylation

Answer 44

- 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

Question 45

SH2 protein binding domain

Answer 45

- 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

Question 46

SH2 affinity

Answer 46

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

Question 47

protein-protein interaction domains

Answer 47

- 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)

Question 48

erythrogenesis

Answer 48

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

Question 49

target genes of STAT TFs

Answer 49

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

Question 50

mutations in EpoR

Answer 50

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

Question 51

lethal effects of erythrogenesis

Answer 51

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

Question 52

Effects of elevated hematocrit (or RBC count)

Answer 52

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

Question 53

use of Epo with athletes

Answer 53

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

Question 54

short term mechanisms that turn off the JAK/STAT pathway

Answer 54

- 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

Question 55

long term regulation of erythrogenesis

Answer 55

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

Question 56

mutations in EpoR gene

Answer 56

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

Question 57

Receptor tyrosine kinase (RTK) and Ras

Answer 57

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

Question 58

components of RTK and Ras pathway

Answer 58

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

Question 59

RTK signal binding and receptor dimerization

Answer 59

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

Question 60

RTK phosphorylation of docking sites

Answer 60

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

Question 61

adaptor proteins binding P-tyr at docking sites

Answer 61

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

Question 62

scaffold proteins

Answer 62

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

Question 63

GRB2

Answer 63

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

Question 64

SH3 protein interaction domain

Answer 64

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

Question 65

GTP bound G-protein in the active conformation

Answer 65

- 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

Question 66

G-protein or GTPase switch protein regulation

Answer 66

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

Question 67

GDP-bound "OFF" state

Answer 67

- 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

Question 68

GEF

Answer 68

- 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

Question 69

GAP

Answer 69

- 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

Question 70

GDI

Answer 70

- inactivated by GDI - guanine nucleotide dissociation inhibitor - increasing the affinity of the nucleotide-binding pocket for GDP, keeping the G-protein inactive or "OFF"

Question 71

Ras-GDP/GTP cycle

Answer 71

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

Question 72

SOS GEF

Answer 72

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

Question 73

conformation states for Ras protein

Answer 73

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

Question 74

GAP for Ras: NF1 (neurofibromatosis)

Answer 74

- 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

Question 75

position of Ras in the pathway

Answer 75

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

Question 76

Adding EGF (control)

Answer 76

- 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

Question 77

adding antibody to Ras

Answer 77

- 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

Question 78

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

Answer 78

-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

Question 79

failure in GTPase activity in Ras

Answer 79

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

Question 80

Failure in GAP protein

Answer 80

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

Question 81

Her2

Answer 81

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

Question 82

Ras activation leading to Raf activation

Answer 82

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

Question 83

Raf

Answer 83

- 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

Question 84

MEK

Answer 84

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

Question 85

MAPK

Answer 85

- 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

Question 86

Activation lip

Answer 86

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

Question 87

activation of transcription

Answer 87

- 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

Question 88

c-fos

Answer 88

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

Question 89

cellular responses

Answer 89

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

Question 90

amplification of the signal

Answer 90

- 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

Question 91

GPCR structure

Answer 91

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

Question 92

Adrenergic stress response

Answer 92

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

Question 93

catecholamines

Answer 93

- 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

Question 94

GPCR catecholamine receptor

Answer 94

-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

Question 95

beta adrenergic receptors

Answer 95

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

Question 96

alpha2 adrenergic receptors

Answer 96

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

Question 97

energy during stress response

Answer 97

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

Question 98

membrane associated proteins

Answer 98

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

Question 99

activation of receptors

Answer 99

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

Question 100

activation of effector AC

Answer 100

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

Question 101

Beta adrenergic receptors mechanism

Answer 101

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

Question 102

alpha2 adrenergic receptor mechanism

Answer 102

- 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

Question 103

activation and inhibition of AC

Answer 103

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

Question 104

Adenylyl cyclase enzyme

Answer 104

-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

Question 105

degradation of cAMP by PDE

Answer 105

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 [ ]

Question 106

secondary messengers

Answer 106

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

Question 107

cAMP role as secondary messenger

Answer 107

responds to the GPCR signalling pathway | the [ ] of cAMP modulates the activity of target proteins including pKA

Question 108

pKA

Answer 108

cAMP -dependent protein kinases = protein kinase A | -pKA is a serine-threoinine kinase that phosphorylates a variety of target proteins

Question 109

activation of pKA

Answer 109

-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

Question 110

regulation of pKA

Answer 110

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

Question 111

how pKA regulation relates to stress response

Answer 111

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

Question 112

targets of pKA

Answer 112

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

Question 113

net effect in muscle to epinephrine

Answer 113

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

Question 114

net effect in liver to epinephrine

Answer 114

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

Question 115

activation of gene transcription

Answer 115

- 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

Question 116

amplification of the signal

Answer 116

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