Signal transduction

Question 1

What are the two main types of kinases?

Answer 1

• Tyrosine kinase

* Serine/threonine kinase

Question 2

What are the two types of GTP binding proteins?

Answer 2

• Trimeric G proteins

* Monomeric GTPases

Question 3

Describe how G protein coupled receptors work

Answer 3

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 4

Describe the structure of G protein coupled receptors

Answer 4

All G protein coupled receptors hace 7 membrane spanning regions with their amino termini on the extracellular face and their carboxy termini on their cytoplasmic face of the plasma membrane
• Many cell surface receptors are coupled to trimeric signal transducing G proteins that bind to either GDP or GTP

Question 5

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

Answer 5

• Binding of a ligand to the receptor changes the conformation
• The receptor binds to Gα protein
• GDP becomes displaced and GTP binds to Gα
• This triggers Gβγ dissociation, activating downstream pathways
• GTP on Gα hydrolyses to GDP in seconds
• Re-association of Gα with Gβγ and the inactivation of adenylate cyclase

Question 6

What is needed by a system

Answer 6

• Receptor
• A transducer (G protein)
• An amplifier (adenylate cyclase) that generates large amounts of second messenger

Question 7

How many types of Gα subunit are there?

Answer 7

20

Question 8

Give 3 examples of Gα subunit

Answer 8

• Gαq (Gq) Stimulates phospholipase C
• Gs: Stimulates adenylate cyclase, increase in cAMP
• Gi: Inhibits adenylate cyclase, decrease in cAMP

Question 9

What are phospholipase isoforms?

Answer 9

Proteins which possess distinct domain structures but catalyse the same reaction (liberation of IP3 and DAG from PIP2)

Question 10

What is the resting calcium level?

Answer 10

~100nM

Question 11

What is active calcium level?

Answer 11

0.5-1 μm

Question 12

What is protein kinase C?

Answer 12

• At least 12 isoforms
• Most are catalytically inactive, soluble proteins in the cytoplasm
• Phosphorylates a wide variety of substate proteins on the threonine and serine residues
• Has substrates in the cytoplasm and some can translocate to the nucleus to phosphorylate nuclear proteins

Question 13

Describe how PKC is activated

Answer 13

• A rise in cytosolic calcium causes PKC to bind to the cytosolic leaflet of the plasma membrane
• One bound, it can be activated by the membrane associated DAG and/or Ca2+

Question 14

Which receptor does adrenaline bind to?

Answer 14

β2 adrenergic receptor

Question 15

Describe the effect of adrenaline binding to the receptor

Answer 15

• Intracellular concentration of cAMP is increased as the receptor couples to Gas
• cAMP is synthesised within cells from aTP by the enzyme adenylate cyclase
• cAMP is degraded by the enzyme cAMP phosphodiesterase

Question 16

What does signal transduction result in?

Answer 16

An amplification of a signal

Question 17

How can PKC act in the nucleus?

Answer 17

• PKA can activate transcription of genes containing the cAMP response elements of CREs in their promoter
• CREB binds to this sequence and activates transcription of downstream genes
• When CREB is unphosphorylated it is inactive, only when it is phosphorylated can it activate transcription

Question 18

What is the cholera toxin?

Answer 18

• Oligomeric complex which after cleavage becomes active and enters the intestinal epithelial cells to stimulate Gas
• Causes an over production of cAMP
• 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, causing dehydration

Question 19

How does the pertussis toxin act?

Answer 19

• Inhibits Gi, leading to an overproduction of cAMP in th lung epithelia

Question 20

What activates RTKs to control cell proliferation?

Answer 20

Insulin like growth factors

Question 21

Describe how RTKs are activated

Answer 21

• Signal molecule in a dimer form binds
• Kinase activity is stimulated
• Tyrosine is phosphorylated
• Signal is relayed by activated signalling proteins into the cells interior (bound to phosphorylated tyrosine)

Question 22

Describe how RTKs activate Ras

Answer 22

• Adaptor proteins bind to the phosphorylated tyrosine
• Causes Ras activating protein to activate Ras
• GDP dissociates and GTP binds, activating Ras

Question 23

What does Ras do?

Answer 23

Regulates cellular proteins:
• Proliferation
• Cytoskeletal dynamics
• Membrane trafficking/vesicular transport \

Question 24

What targets Rac/Rho subgroups of Ras?

Answer 24

Clostridial cytotoxins

Question 25

Cancer and Ras

Answer 25

Ras mutated to be constantly bound to GTP in 20% of cancers

Question 26

What does Ras activate? and what does this cause?

Answer 26

The MAPK pathway
• Changes in protein activity
• Changes in gene expression

Question 27

What is the EGFR receptor pathway?

Answer 27

• 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).

Question 28

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

Answer 28

Tumourgenesis

These cause overexpression and/or hyperactivation of the respective proteins