Signal Transduction

Question 1

Why do we need signal transduction?

Answer 1

As most things that affect cell activity or function do not enter cells Instead they act on membrane-bound receptors that control signalling proteins via the production of secondary messengers

Question 2

What is the function of secondary messengers ?

Answer 2

They mediate cell activity (including gene expression)

Question 3

What are the 2 ways that a signal can be transduced ?

Answer 3

1). Via proteins inside the cell which can change its behaviour (fast response - Seconds to Minutes) 2). Via making new proteins by transcription in order to change a cells behaviour (Slow - Minutes to Hours)

Question 4

How do signalling proteins generally act ?

Answer 4

Many signalling proteins act as molecular switches

Question 5

How many kinases and phosphates does the human genome encode?

Answer 5

Approx 520 kinases and Approx 150 phosphates

Question 6

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

Answer 6

1). Signalling via phosphorylation; - The signal goes into the cell via the receptor and activates protein kinase which coverts ATP to ADP and adds a phosphate onto the target protein to activate it - The phosphate is cleaved off the activated protein using protein phosphatase and this releases phosphate so it can bind back onto ADP to form ATP and the protein is deactivated 1). Signalling via GTP binding; - The signal goes into the cell via the receptor and activates GTP binding which coverts GDP to GTP which binds onto the target protein to activate it - The GTP is attached to the activated protein is hydrolysed using GTP hydrolysis which releases a phosphate, consequently leaving GDP binded to the protein and the protein is deactivated

Question 7

What are the two main types of kinases ?

Answer 7

Tyrosine kinase and Serine/Threonine kinase

Question 8

What are the two main types of GTP-binding proteins ?

Answer 8

Trimeric G proteins and Monomeric GTPases

Question 9

What does ligand binding do ?

Answer 9

• Ligand binding activates a G-protein which in turn activates or inhibits another protein, which is often an enzyme which generates a specific second messenger

Question 10

What does GPCR’s stand for?

Answer 10

G-protein coupled receptors

Question 11

What series of steps occurs when activating G-protein-coupled receptors?

Answer 11

• An extracellular ligand binds to then G-protein-coupled receptor protein found in the plasma membrane
• This activates the inactive G-signal transducing protein (this is known as the G-protein activation) which inhibits/activates a down stream inactivated effector enzyme
• An inactivated effector enzyme (like adenylyl cyclase or phospholipase c) binds onto the now activated form of G-protein (this is known as complex formation)
• The Active effector (G-protein + Enzyme) generates “second messengers” (like cAMP; inositol 1,4,5-triphosphate; 1,2 - diacylglycerol)

Question 12

What do all G-protein-coupled receptors have?

Answer 12

7 membrane spanning regions; - with their amino termini (N-terminus) facing extracellularly - with their carboxyl termini (C-terminus) facing intracellularly from the plasma membrane

Question 13

How can many of the cell-surface receptors be described?

Answer 13

Many of the cell-surface receptors are coupled to trimeric signal-transducing proteins (composed of three different subunits - Alpha, Beta and Gamma) that bind to either GTP or GDP

Question 14

Describe the mechanisms of action of G-protein signal transduction?

Answer 14

• The ligand binds to the receptor and changes its conformation, causing the receptor to bind to the G-alpha (Ga) protein, during this GDP is displaced by GTP which binds to the G-alpha (Ga) protein
• This triggers G-Beta-Gamma (GBy) to release from the Gs trimeric protein (Containing G-alpha protein and G-Beta-Gamma protein joined together)
• This release of G-Beta-Gamma (GBy) activates downstream pathways
• These pathways only last for a short period of time as GTP which is bonded to G-alpha hydrolyses back to GDP in seconds, causing G-Beta-Gamma (GBy) to bind back onto G-alpha and the inactivation of the enzyme adenylate cyclase

Question 15

What is GTP required for?

Answer 15

GTP is required for the ligand-induced stimulation of Adenylate Cyclase (enzyme)

Question 16

What couples to G-alpha ?

Answer 16

Glucagon

Question 17

What are the 3 things that is required for a signal transduction system?

Answer 17

1). A receptor (e.g - Glucagon) 2). A transducer (G-protein) 3). An amplifier (adenylate cyclase) which generates a large amount of second messenger

Question 18

What is the role of Adenylate cyclase ?

Answer 18

Amplifies signals and produces large amounts of second messengers

Question 19

What is the structure of G-protein-coupled receptors (GPCR) ?

Answer 19

Their receptors consists of 7 transmembrane helices (hydrophobic amino acids) which sit in the plasma membrane - which are Beta helices (called Beta 2 adrenergic receptor) They interact with heterotrimeric G-protein complex on the extracellular side (has Alpha, Beta and Gamma subunits)

Question 20

How many types of G-alpha subunits is there?

Answer 20

20

Question 21

What are the 3 main types of G-alpha classes and their functions?

Answer 21

Gaq (or Gq) - Stimulates phospholipase C Gs - Stimulates adenylate cyclase, increases cAMP Gi - Inhibits adenylate cyclase, decreases cAMP

Question 22

What is unique about all GPCR’s (G-protein-coupled receptors) ?

Answer 22

All GPCR’s exhibit preferential association with a particular G-protein (or subset of G-protein) GCPRs signal via GTP-mediated activation of G proteins

Question 23

How do GPCR’s signal and draw a diagram ?

Answer 23

• A signalling molecule activates a G-protein linked receptor which activates a G-protein subunit
• This activates the enzyme phospholipase C which liberates two signalling molecules from Phosphatidylinositol 4,5-biphosphate (PIP2), a phospholipid found in the lipid bilayer
• This creates inositol 1,4,5 triphosphate (IP3) and diacyglycerol (DAG)
• The inositol 1,4,5 triphosphate (IP3) activates the Calcium channel on the endoplasmic reticulum causing a calcium influx inside the cell to activate PKC (Protein kinase C) and the binding of the diacyglycerol (DAG) also further activates the Protein kinase C (both substances are required to activate it)

Question 24

What are phospholipase C isoforms ?

Answer 24

Phospholipase C isoforms are proteins which possess distinct domain structures but catalyse the same reaction (releasing of IP3 and DAG from PIP2) Some domains are common (catalytic, membrane localisation) and some are unique (regulatory)

Question 25

How would you describe Cytosolic Calcium levels?

Answer 25

Cytosolic Calcium levels are dynamic; - Calcium can enter from intracellular stores or from outside the cell via calcium channels (receptor or voltage operated) - Calcium can exit via other channels present in the plasma or organelle membranes

Question 26

What is the resting and activated calcium concentrations?

Answer 26

Resting calcium concentration - about 100nM Activated calcium concentration - 0.5 - 1uM

Question 27

What is cellular response dependent on?

Answer 27

The duration of the signal

Question 28

Give an example of a biological process that Calcium signals trigger?

Answer 28

Fertilisation of an egg by a sperm - initial spark by PLC-Z (Phospholipase C Zeta) triggers opening of surface calcium channels The calcium wave triggers the start of embryonic development and prevents other sperm from entering the cell

Question 29

What are Protein Kinase C (PKC) properties?

Answer 29

PKC's are a large family with at least 12 different isoforms Most are catalytically inactive, soluble proteins in the cytoplasm

Question 30

What does a rise in cytosolic calcium levels cause PKC (Protein kinase C) to do?

Answer 30

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

Question 31

What do PKC's substrates in the cytoplasm and some isoforms do?

Answer 31

PKC has substrates in the cytoplasm and some isoforms which can translocate to the nucleus to phosphorylate nuclear proteins, and thus function in a transient (short lasting) way or in a more permanent way (in gene transcription)

Question 32

What can "indirectly' alter gene expression?

Answer 32

Protein Kinase C activation

Question 33

What is another common example of GPCR signalling ?

Answer 33

Binding of adrenalin to the B2 adrenergic receptor

Question 34

How does the Binding of adrenalin to the B2 adrenergic receptor affect the body?

Answer 34

It mediates the body's response to stress/fear (fight or flight system) - Release of Glucose and Fatty acids from liver/fat cells - Increased concentration of Cardiac muscle

Question 35

How does Adrenalin binding to the B2 adrenergic receptor chemically work?

Answer 35

The binding of Adrenalin to B2 adrenergic receptor increases the intracellular concentration of cAMP (cyclic AMP) as receptor couples to G-alpha S

Question 36

Where is cAMP synthesised and by what enzyme?

Answer 36

cAMP is synthesised within cells from ATP by the Adenylate cyclase - Different receptors use the same Adenylate cyclase enzyme (each receptor does have its own designated Adenylate cyclase)

Question 37

What is cAMP degraded by ?

Answer 37

cAMP is degraded by the enzyme cAMP phosphodiesterase

Question 38

How does an increase cAMP activate the cAMP-dependent protein kinase ?

Answer 38

cAMP binds to the regulatory subunits found in the cAMP-dependent protein kinase and causes the catalytic subunits to detach at the pseudosubstrate site, allowing the substrate site to be free to bind to cAMP

Question 39

What does signal transduction cause?

Answer 39

Amplification of a signal - A small change at the top of the chain can have a large change at the bottom (1 epinephrine gives 5 products)

Question 40

What can the catalytic subunit of PKA (Protein-kinase a) phosphorylate?

Answer 40

The catalytic subunit of PKA (Protein-kinase a) can phosphorylate substrates on Serine and Threonine. It has substrates in the membrane, cytoplasm and nucleus

Question 41

What is the role of PKA in the nucleus ?

Answer 41

- PKA in the nucleus can activate transcription of genes containing cAMP response elements (CREs) in their promoter.

Question 42

What is CREB?

Answer 42

CREB (cAMP response element binding) protein is a specific transcription factor which binds to the sequence of genes and activates transcription of downstream genes

Question 43

How is CREB activated and inactivated ?

Answer 43

When CREB is unphosphorylated it is inactive When CREB is phosphorylated it does CREB (cAMP response element binding) active transcription

Question 44

What is Cholera Toxin ?

Answer 44

Cholera toxin is an oligomeric complex (has repeating units of monomers) which after cleavage becomes active and enters the intestinal epithelial cells to stimulate G-alpha S The over stimulation of cAMP production results in a release of water and ions including Sodium, Potassium, Chlorine and Bicarbonate ions into the lumen of the small intestine This leads to rapid fluid loss and dehydration

Question 45

What is Pertussis Toxin ?

Answer 45

Pertussis Toxin works in a reverse manner from Cholera toxin and it inhibits G-alpha-i to increase cAMP production in lung epithelia causing whooping cough

Question 46

Explain what happens in the enzyme-linked receptor; Receptor Tyrosine Kinases (RTK's)

Answer 46

1) . The Receptor Tyrosine Kinases (RTK's) are inactive and apart from one another 2) . A signalling molecule in the form of a dimer (An oligomer made of 2 monomers joined together) binds to the 2 RTK's, consequentially brining them close to one another 3) . Due to how close the 2 RTK's are their tyrosine kinase domains are touching one another and are stimulating kinase activity 4) . These kinases are activated and add phosphate groups to the tyrosine amino acids 5) . Intracellular signalling proteins bind to the phosphorylated tyrosine. This causes a signal relayed by activating signalling proteins into the cells interior

Question 47

Give an example of a Receptor Tyrosine Kinases (RTK's) response?

Answer 47

Insulin-like growth factors activate RTK's to control cell production

Question 48

What do Receptor Tyrosine Kinases (RTK's) activate?

Answer 48

A small GTPase called 'Ras'

Question 49

How does the activated RTK activate Ras?

Answer 49

The activated RTK has an adaptor protein bind to one of the phosphate groups on the tyrosine. The adaptor protein has a Ras-activating protein attached to it which consequently activates Ras by displacing the GDP attached to it (making it inactive) with GTP (making it activated). The Ras protein sends a transmission of signal onwards

Question 50

What is the function of Ras?

Answer 50

Ras regulates cellular processes like; - Proliferation - Cytoskeletal dynamics - Membrane trafficking/vesicular transport

Question 51

How can GTPases cause disease?

Answer 51

If GTPases are damaged it can have catastrophic consequences for the cell and organism Several small GTPases of the Rac/Rho subfamily are direct targets for clostridial cytotoxins Ras proteins are mutated to become in their almost constantly active (GTP-bound) form in approximately 20% of human cancers

Question 52

What does Ras activate and how does it do this?

Answer 52

Ras activates the MAPK (Mitogen-activated protein kinase pathway) 1). The active Ras protein sends of signals to activate MAP-kinase-kinase-kinase which uses 1 kinase to remove a phosphate group of ATP changing it into ADP and transfer it to the MAP leaving us with MAP-kinase-kinase with a phosphate group attached 2). The MAP-Kinase-Kinase sends off a signal to cause another of its kinases to cleave ATP into ADP and attack the phosphate group to the now MAP-kinase with 2 phosphates attached 3) The MAP-Kinase sends off a signal to cause another of its kinases to cleave ATP into ADP and attack the phosphate group to the now MAP with 3 phosphates attached 4). The phosphates can bind to protein X or protein Y to change protein activities inside the cell or The phosphates can bind to gene regulatory A or B to change the cells gene expression

Question 53

What is the EGFR receptor activated by and what does it cause?

Answer 53

The EGFR (epidermal growth factor receptor) is a RTK (Receptor Tyrosine Kinase) which is activated by TGF-alpha (Transforming growth factor alpha) EGFR activates Ras via Grb/SOS proteins Ras activates Raf (kinase) which stimulates gene transcription via other kinases (MEK and ERK)

Question 54

What occurs when there are mutations in the EGFR (epidermal growth factor receptor)

Answer 54

Mutations in the EGFR (epidermal growth factor receptor) Ras and Raf are associated with tumorigenesis (the production of formation of tumours) These cause an over expression and/or hyper activation of the respective proteins

Question 55

How do signalling pathways interact ?

Answer 55

Signalling pathways are all interconnected and multiple pathways end up in the same place and one can affect another They all generally activate gene regulatory proteins or target proteins