Cell Signalling

Question 1

Describe how cell activity is affected.

Answer 1

• Most things which affect cell activity or function do not enter the cells.
• They act on membrane-bound receptors that control signalling proteins via the production of second messengers.
• These mediate cell activity.

Question 2

What are the general principles of signal trasduction?

Answer 2

• Many signalling proteins act as molecular switches.
• There are two common ways to activate / deactivate signalling proteins.
• Human genomes encodes ~520 kinases and ~150 phosphatases.
• There are two main types of kinases.
• There are two types of GTP-binding proteins.

Question 3

What are the 2 main ways of switching signalling proteins on/off?

Answer 3

• Phosphorylation
• GDP binding

Question 4

What are the 2 main types of kinases?

Answer 4

• Tyrosine kinase
• Serine / threonine kinase

Question 5

What are the 2 types of GTP-binding proteins?

Answer 5

• Trimeric G proteins
• Monomeric GTPases

Question 6

Explain the role of G-protein-coupled receptors in signal transduction.

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

What is a G-protein-coupled receptor?

Answer 7

• 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.
  
  • The 7 membrane-spanning regions are alpha helices.
• A 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.
• Trimeric: composed of 3 different subunits (α, β and Ɣ)

Question 8

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

Answer 8

• Binding of the ligand to the receptor changes its conformation, causing it to bind to the Gα protein in such a way that GDP is displaces and GTP is bound.
• This triggers Gβɣ dissociation activating downstream pathways.
• Activation is short-lived, as GTP bound to Gα hydrolyses to GDP in seconds, leading to the re-association of Gα with Gβɣ and inactivation of adenylate cyclase.

Question 9

Describe how adenylate cyclase is stimulated.

Answer 9

• GTP is required for the ligand-induced stimulation of adenylate cyclase.
• Glucagon receptor couples to Gα_S.
• Overall, the system needs:
  
  • A receptor
  • A transducer (G-protein)
  • An amplifier (adenylate cyclase) that generates large amounts of a second messenger.
    
    • cAMP is the second messenger made by adenylate cyclase.

Question 10

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

Answer 10

• Receptors consist of 7 transmembrane helices (hydrophobic amino acids) that reside in the plasma membrane.
• Interacts with heterotrimeric G-protein complex on the extracellular side.

Question 11

Which type of G-protein stimulates phospholipase C?

Answer 11

Gα_q

Question 12

Which type of G-protein stimulates adenylate cyclase and increases cAMP?

Answer 12

G_s

Question 13

Which type of G-protein inhibits adenylate cyclase and decreases cAMP?

Answer 13

G_i

Question 14

What are the functions of the Gβ/ɣ dimer?

Answer 14

• Gate ion channels
• Stimulates adenylate cyclase
• Stimulates PLA₂
• Stimulates PLC-β, PLC-ε and PLC-𝜼

Question 15

How does phospholipase C become activated?

Answer 15

Its substrate is phospholipis which resides in the cell membrane.

1-2% of the cell membrane is this phospholipid so it is not abundant.

Question 16

What are phospholipase C isoforms?

Answer 16

• 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 uique (regulatory).
• Acivated by different pathways.

Question 17

Describe calcium signalling.

Answer 17

• Cytosolic calcium levels are dynamic.
• 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 18

What is the resting calcium concentration of a cell?

Answer 18

~100nM

Question 19

What is the activated calcium concentration of a cell?

Answer 19

0.5-1µM.

Cellular response depends upon the duration of the signal.

Question 20

What is the effect of the calcium wave on an egg cell?

Answer 20

• Calcium wave does 2 things:
  
  • Tells the cell to start dividing
  • Prevents any more sperm coming in because an egg should not be fertilised

Question 21

Describe protein kinase C.

Answer 21

• A large family: at least 12 different isoforms.
• Most are present as catalytically inactive, soluble proteins in the cytoplasm.

Question 22

What happens to PKC in the cell?

Answer 22

• 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+.
• PKC then phosphorylates

Question 23

Protein kinase C functions ‘indirectly’ to do what?

Answer 23

To alter gene expression.

The genes can phosphorylate kinases.

Question 24

Binding of adrenalin to the β₂ adrenergic receptor is another type ot G-protein-coupled receptor signalling.

Describe what happens.

Answer 24

• 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
• Bindind of adrenaline to β₂ adrenergic receptor increases the intracellular concentration of cAMP (cyclic AMP) as receptor couples to Gαs.
• cAMP is synthesised within cells from ATP by the enzyme adenylate cyclase.
• cAMP is degraded by the enzyme cAMP phosphodiesterase.
• Different receptors utilise a common adenylate cyclase (ie each receptor does not have its own intrinsic adenylate cyclase).

Question 25

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

Answer 25

Question 26

Describe the effect of signal transduction.

Answer 26

• Signal transduction results in an amplification of a signal:
  
  • A small change at the top can have a large change at the bottom.
• A large amount of product is made relative to the amount of reactants. A small amount of signal can generate a large amount of second messenger within the cell.

Question 27

What can the catalytic subunits of PKA do?

Answer 27

The catalytic subunits of PKA can phosphorylate sibstrates on serine or threonine residues. It has substrates in the membrane, cytoplasm and the nucleus.

Question 28

What does PKA do in the nucleus?

Answer 28

• In the nucleus, 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.

Question 29

What is the function of CREB?

Answer 29

• 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.

Question 30

Describe mechanism of action of the cholera toxin.

Answer 30

• 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 intestine.
• This leads to rapid fluid loss and dehydration.

Question 31

What are receptor tyrosine kinases (RTKs)?

Answer 31

• High affinity cell surface receptors for many polypeptide growth factors, cytokines and hormones.
• Enzyme-linked receptor.
• Example: insulin-like growth factors activate RTKs to control cell proliferation.
• RTKs are associated with cancer because they control cell proliferation - in cancer there is often uncontrolled cell proliferation.

Question 32

What is Ras?

Answer 32

• Ras superfamily contains over 100 members: Rho, Rap, Rab, Arf etc.
• They regulate cellular processes: proliferation, cytoskeletal dynamics, membrane trafficking / vesicular transport.

Question 33

What is the role of GTPases in disease?

Answer 33

• Damage to 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.
• They bind GTP and cannot release / hydrolyse it again. This is associated with cancerous cells.

Question 34

What does Ras activate?

Answer 34

• The mitogen-activated protein kinase (MAPK) pathway.
• Ras activates Raf (kinase), which in turn stimulates gene transcription via other kinases (MEK and ERK).

Question 35

What is EGFR?

Answer 35

EGFR (epidermal growth factor receptor) is an RTK (receptor tyrosine kinase) activated by TGFα (transforming growth factor alpha).

Question 36

What does the EGFR pathway do?

Answer 36

The EGFR activates Ras via Grb2/SOS proteins.

Question 37

What is associated with mutations in EGFR, Ras and Raf?

Answer 37

Tumorigenesis because they cause overexpression and/or hyperactivation of the respective proteins.

Question 38

FOCUS ON THESE PATHWAYS FOR THE EXAM

Question 39

How does the pertussis toxin work?

Answer 39

• Pertussis toxin works in the reverse manner to the cholera toxin.
• It Inhibits Gαi in order to increase cAMP production in lung epithelia.
• Pertussis toxin - whooping cough.