Signal Transduction

Question 1

What do many signalling proteins also act as?

Answer 1

molecular switches

Question 2

What are the two common ways to activate/deactivate signalling proteins?

Answer 2

By phosphorylation

By GTP binding

Question 3

How many kinases/phosphatases does the human genome code for?

Answer 3

~520
kinases and

~150 phosphatases

Question 4

What are the two main kinds of kinases?

Answer 4

• tyrosine kinase

- serine/threonine kinase

Question 5

What are the two types of GTP-binding proteins?

Answer 5

• Trimeric G proteins

- Monomeric GTPases

Question 6

What process occurs at G-protein coupled receptors (GPCRs)?

Answer 6

Ligand binding activates a G-protein which in turn activates or inhibits another protein. Often this is an enzyme that generates a specific second messenger.

Question 7

How is an intracellular second message generated?

Answer 7

Ligand binding to a G protein coupled receptor activates the associated G protein which in turn inhibits / activates a downstream enzyme to generate an intracellular second message. G protein activation and complex formation are part of a cycle.

Question 8

Describe the structure of a G-protein-coupled receptor?

Answer 8

All G-protein coupled receptors have 7 membrane spanning regions with their amino termini on the extracellular face and their carboxy termini on the cytoplasmic face of the plasma membrane.

Question 9

What can trimeric signal transducing G proteins bind?

Answer 9

Many cell-surface receptors are coupled to trimeric signal-transducing G proteins that bind either GTP or GDP

Question 10

What does trimeric mean?

Answer 10

composed of three different subunits (a, b and gamma in G proteins - beta and gamma split off from alpha to activate it)

Question 11

What is the mechanism of action of a trimeric G protein in a G protein coupled receptor?

Answer 11

Binding of the ligand to the receptor changes its conformation, causing it to bind to the G(alpha) protein in such a way that GDP is displaced and GTP is bound.

This triggers G(b+g) dissociation activating downstream pathways.

Activation is short-lived, as GTP bound to G(alpha) hydrolyzes to GDP in seconds, leading to the re-association of Ga with Gbg and inactivation of adenylate cyclase.

Question 12

What is GTP required for as discovered by Martin Rodbell?

Answer 12

GTP is required for the ligand-induced stimulation of adenylate cyclase.

Question 13

What does the glucagon receptor bind to?

Answer 13

Glucagon receptor couples to Gα(small)s

Question 14

Overall, what does a signal transduction system need?

Answer 14

A receptor

A transducer (G-protein) and

An amplifier (adenylate cyclase) that generates large amounts of a second messenger.

Question 15

What is GPCR structure?

Answer 15

Receptors consist of 7 TM helices (hydrophobic amino acids) that reside in the plasma membrane

Interacts with heterotrimeric G protein complex on the extracellular side

Question 16

What part of GPCR crosses the plasma membrane?

Answer 16

The receptor, eg;

β2 adrenergic receptor
7TM receptor

Question 17

What part of GPCR is the G protein signalling complex?

Answer 17

α, β and γ subunits

Question 18

How many types of G proteins are there?

Answer 18

20

Question 19

What are all the types of G proteins?

Answer 19

(All not greek script is subscript)
Gαq (or Gq) 
Gs 
Gi
G12/13 
Golf 
Gt 
Gβ/γ dimer

Question 20

What are the important three G protein types and what do they do?

Answer 20

Gαq (or Gq) → stimulates phospholipase C
Gs → stimulates adenylate cyclase, increases cAMP
Gi → inhibits adenylate cyclase, decreases cAMP

Question 21

What does Gαq (or Gq) do?

Answer 21

stimulates phospholipase C

Question 22

What does Gs do?

Answer 22

stimulates adenylate cyclase, increases cAMP

Question 23

What does Gi do?

Answer 23

inhibits adenylate cyclase, decreases cAMP

Question 24

What does G12/13 do?

Answer 24

regulates cytoskeleton, cell junctions, movement, stimulates PLC-ε

Question 25

What does Golf do?

Answer 25

activates olfactory receptors

Question 26

What does Gt do?

Answer 26

transduces visual signals with rhodopsin in the retina

Question 27

What does a Gβ/γ dimer do?

Answer 27

→ gate ion channels → stimulates PLA2 → stimulates adenylate cyclase → stimulates PLC-β, PLC-ε and PLC-η

Question 28

How many Gβ’s and Gγ’s are there?

Answer 28

6 Gβ’s and 12 Gγ’s | 72 combinations

Question 29

What do all GPCRs exhibit?

Answer 29

preferential association with a particular G protein | or subset of G proteins

Question 30

What does GPCR stand for?

Answer 30

G protein-coupled receptor

Question 31

What are phospholipase C isoforms?

Answer 31

Proteins which possess distinct domain structures but catalyse the same reaction (liberation of IP3 and DAG from PIP2) Some domains are common (catalytic, membrane localisation), Some domains are unique (regulatory)

Question 32

How are phospholipase C isoforms activated

Answer 32

``` Different pathways including GPCR activation (Gαq) Receptor tyrosine kinases Kinases (possibly ↑Ca2+) Small GTPases Fertilisation ↑Ca2+, Gβγ, other mechanisms? ```

Question 33

What is the normal calcium level found in the cytosol?

Answer 33

Trick question bitchboy: cytosolic calcium levels are dynamic

Question 34

How does calcium enter/exit the cytosol?

Answer 34

Calcium can enter from intracellular stores or from outside the cell via calcium channels Channels may be receptor or voltage operated. Calcium can exit via other channels present in plasma or organelle membranes

Question 35

What is the resting calcium concentration in the cytosol?

Answer 35

~100 nM

Question 36

What is the activated calcium concentration in the cytosol?

Answer 36

Activated calcium concentration = 0.5-1 μM

Question 37

Why is the activated cytosolic calcium concentration variable?

Answer 37

Cellular response depends upon duration of the signal

Question 38

What biological processes can calcium signals trigger

Answer 38

Many, an example: fertilisation of an egg by a sperm – initial spark by PLC-ζ (zeta) triggers opening of surface calcium channels Calcium wave triggers start of embryonic development and prevents other sperm from entering the cell

Question 39

How many different isoforms does protein kinase C have?

Answer 39

At least 12 probably

Question 40

How are most protein kinase C-s (PKCs) found in the cell

Answer 40

Most are present as catalytically inactive, soluble proteins in the cytoplasm

Question 41

What causes the activation of protein kinase C?

Answer 41

Rise in cytosolic calcium levels causes PKC to bind to the cytosolic leaflet of the plasma membrane, where it can be activated by the membrane-associated DAG and/or Ca2+.

Question 42

What does activated protein kinase C do?

Answer 42

PKC then phosphorylates a wide variety of substrate proteins on serine and threonine residues.

Question 43

What other functions does protein kinase C have?

Answer 43

PKC has substrates in the cytoplasm and some isoforms can translocate to the nucleus to phosphorylate nuclear proteins, and can thus function in a transient way or in a more permanent way (gene transcription). Protein kinase C activation can also function “indirectly” to alter gene expression

Question 44

Provide an example of other types of GPCR signalling

Answer 44

Mediates the body’s response to stress/fear (fight or flight!!)  release of glucose and fatty acids from liver/fat cells  increased contraction of cardiac muscle Binding of adrenalin to b2 adrenergic receptor increases the intracellular concentration of cAMP (cyclic AMP) as receptor couples to Gs cAMP is synthesized within cells from ATP by the enzyme adenylate cyclase cAMP is degraded by the enzyme cAMP phosphodiesterase Different receptors utilize a common adenylate cyclase (i.e., each receptor does not have its own intrinsic adenylate cyclase).

Question 45

How does increased cAMP activate the cAMP-dependent protein kinase, PKA?

Answer 45

cAMP binds to regulatory subunits bound to catalytic subunits, causing a conformational change which releases the catalytic subunits, thus exposing the catalytic site

Question 46

What does signal transduction do to a signal?

Answer 46

It amplifies it

Question 47

What can the catalytic subunit of PKA phosphorylate?

Answer 47

substrates on serine or threonine residues

Question 48

Where does PKA have substrates?

Answer 48

in the membrane, cytoplasm and the nucleus.

Question 49

What does PKA do in the nucleus?

Answer 49

PKA can activate transcription of genes containing cAMP response elements, or CREs in their promoter. A specific transcription factor, the cAMP response element binding protein, CREB, binds to this sequence and activates transcription of downstream genes. When CREB is unphosphorylated, it is inactive; only in its phosphorylated state does CREB activate transcription. i.e. ligand binding to a cell surface receptor can induce gene expression

Question 50

What is the cholera toxin and what does it do?

Answer 50

Oligomeric complex which after cleavage becomes active and enters intestinal epithelial cells to stimulate Gαs The overstimulation of cAMP production results in a release of water and ions including Na+, K+, Cl- and HCO3- into the lumen of the small intestine This leads to rapid fluid loss and dehydration

Question 51

What does the pertussis toxin do?

Answer 51

Pertussis toxin acts in the reverse manner to cholera for the same effect, inhibiting Gαi to increase cAMP production in lung epithelia

Question 52

Describe the action of receptor tyrosine kinases (RTKs)

Answer 52

They are found across the membrane in an inactive state. A signal molecule in the form of a dimer then binds, stimulating kinase activity. As an active receptor tyrosine kinase they bind phosphorylated tyrosine. Intracellular signalling proteins then bind to phosphorylated tyrosines. Thus, signal is relayed by activating signalling proteins into the cells interior e.g. Insulin-like growth factors activate RTKs to control cell proliferation

Question 53

How long do GPCRs take to have an effect?

Answer 53

Seconds

Question 54

How long do Kinase linked receptors take to have an effect?

Answer 54

Hours

Question 55

What are receptor tyrosine kinases an example of?

Answer 55

Kinase linked receptors

Question 56

What are the roles a kinase linked receptor can fulfil?

Answer 56

Enzyme-linked receptors | Maybe more idk

Question 57

What do Ras proteins do?

Answer 57

Regulate cellular processes: proliferation, cytoskeletal dynamics, membrane trafficking/vesicular transport

Question 58

How do Ras proteins do so goddamn much?

Answer 58

Ras superfamily contains over 100 members: Rho, Rap, Rab, Arf etc.

Question 59

How are Ras proteins activated?

Answer 59

Receptor Tyrosine Kinases activate Ras (Activates adaptor protein which activates Ras-activating protein which replaces GDP bound to Ras with GTP thus activating it and continuing the signal)

Question 60

What are Ras proteins?

Answer 60

GTPases

Question 61

Discuss GTPases and disease

Answer 61

Damage to these small GTPase switches can have catastrophic consequences for the cell and the organism. Several small GTPases of the Rac/Rho subfamily are direct targets for clostridial cytotoxins. Further, Ras proteins are mutated to a constitutively-active (GTP-bound) form in approximately 20% of human cancers.

Question 62

What do Ras proteins activate?

Answer 62

the MAPK (mitogen-activated protein kinase) pathway

Question 63

what does the MAPK (mitogen-activated protein kinase) pathway cause?

Answer 63

Changes in protein activity Changes in gene expression

Question 64

Discuss EGFR pathway and cancer

Answer 64

EGFR (epidermal growth factor receptor) is a RTK activated by TGFα (Transforming growth factor alpha) The receptor activates Ras via Grb2/SOS proteins Ras activates Raf (kinase), which in turn stimulates gene transcription via other kinases (MEK and ERK). Mutations in EGFR, Ras and Raf are associated with tumorigenesis These cause overexpression and/or hyperactivation of the respective proteins

Question 65

What effect does the EGFR receptor pathway have on Ras proteins?

Answer 65

The receptor activates Ras via Grb2/SOS proteins

Question 66

What is the EGDR?

Answer 66

epidermal growth factor receptor

Question 67

Do signalling pathways interconnect?

Answer 67

Hell to the YES

Question 68

Why med?

Answer 68

Fuck else to do