Cross-Talk and Compartmentalisation in Cellular Signalling

Question 1

What type of receptor is the insulin receptor?

Answer 1

A Receptor Tyrosine Kinase (RTK)

Question 2

What is recruited upon ligand binding to the insulin receptor?

Answer 2

IRS-1; multi-docking protein

Question 3

Name a signalling molecule recruited by IRS-1 and describe how it binds to the signalling molecule.

Answer 3

Phosphoinositol-3-kinase (PI3K) is activated upon binding of the SH2 domain in the p85 subunit to the phosphorylated IRS-1

Question 4

What is the role of PI3K?

Answer 4

Catalytic subunit, p110, phosphorylates PI4P or PI(4,5)P2 at the 3-position, synthesising PI(3,4)P2 and PI(3,4,5)P3 respectively.
This creates binding sites for yet other domains, e.g. PH domain that binds the phosphoinositols

Question 5

What is the role of PI(3,4)P2?

Answer 5

PI(3,4)P2 provides a binding site for the PH domain of PKB/Akt, partially activating it by exposing the activation lip allowing it to be phosphorylated/activated by PDPK1, which is co-recruited to the same site.

Question 6

What is the role of PDPK1?

Answer 6

3-Phosphoinositol-dependant protein kinase-1, PDPK1 phosphorylates the activation lip on PKB/Akt that is revealed by PI(3,4)P2; activating PKB/Akt

Question 7

How are PDPK1 and PKB/Akt able to be recruited within close proximity of each other?

Answer 7

PDPK1 and PKB/Akt both have PH domains which bind phosphoinositols, thus PDPK1 is recruited to the same site as PKB/Akt. Recruitment allows activation.

Question 8

What are some cellular effects of PKB activity?

Answer 8

Expression of Fas ligand
Expression of anti-apoptotic genes
Involved in glycogen synthesis, protein synthesis… cAMP modulation.

Question 9

Explain the effect of insulin on blood glucose?

Answer 9

Insulin binding causes PKB/Akt activation through IRS-1 complex, causing phosphorylation and inactivation of glycogen synthase kinase-3 (GSK3).

Inactive GSK3 means glycogen synthase remains in its un-phosphorylated, active state.

Glycogen synthase occurs and blood glucose lowers.

Question 10

When is insulin released?

Answer 10

In response to high blood glucose levels

Question 11

In the case of GS and GSK3, is phosphorylation activating or inactivating?

Answer 11

Inactivating

Question 12

Explain how blood glucose levels rise in the absence of insulin.

Answer 12

Insulin receptor not activated, IRS-1 not recruited and scaffolding protein complex not formed, PI3K is not activated, PDPK1 and PKB/Akt are not co-recruited, PKB/Akt remains inactive so GSK3 remains active, which inhibits GS activity, attenuating glycogen synthesis. Glucose levels rise through cross-talk promotion of degradation.

Question 13

What is the intersection of adrenaline and insulin activity in regulation of blood glucose? (Where do these two pathways meet molecularly?)

Answer 13

Adrenaline leads to activation of PKA which phosphorylates GS, inactivating it. -> Blood glucose levels rise

Insulin leads to activation of PKB/Akt, which allows GS to be dephosphorylated and therefore active. -> Blood glucose levels decline

Question 14

Explain how signalling through adenylyl cyclase is antagonised.

Answer 14

cAMP phosphodiesterase (cAMP-PDE) continually catalyses the hydrolysis of 3’,5’-cAMP -> 5’-AMP. This ensures cAMP only acts when the cell is stimulated.

Question 15

Which proteins are able to regulate phosphodiesterases?

Answer 15

PKA, PKB, Calmodulin… and others.

This allows a further layer of regulation fine-tuning the cAMP response

Question 16

What is Rapamycin?

Answer 16

A fungal metabolite discovered in the 1990s to be inhibiting the kinase TOR (Target Of Rapamycin) in yeast genetics.

It can be used as a drug to inhibit TOR independently of starvation, for immunosuppression, cancer, longevity and psychiatric conditions.

Question 17

What is kinase mTOR?

Answer 17

mammalian Target Of Rapamycin; a phosphoinositol-3-related kinase, a central regulator of cell growth through protein synthesis, autophagy and respiration.

Question 18

What is the function of mTOR in the liver?

Answer 18

mTOR suppresses ketogenesis, gluconeogenesis and amino acid release; suppresses functions associated with low energy/starvation.

Question 19

What is the function of mTOR in the pancreas?

Answer 19

Cause beta cell growth and proliferation improving insulin production.

Question 20

What is the function of mTOR in adipose tissue?

Answer 20

Lipogenesis and adipocyte differentiation

Question 21

What is the function of mTOR in muscle tissue?

Answer 21

Hypertrophy - growing muscle

Question 22

What is the result of overactive, and underactive mTOR?

Answer 22

Overactivation of mTOR -> obesity + associated factors?

Fasting -> Inhibition of mTOR -> no growth, degradation of muscle and other tissues.

Question 23

How could caloric restriction promote longer life in humans?

Answer 23

mTOR normally inhibits autophagy, stem cell function and promotes mRNA translation while caloric restriction leads to a degree of mTOR inhibition.

This results in increased autophagy, increased stem cell function and decreased mRNA translation. Inhibiton of Rapamycin has same effect.

(Shown in animal studies, suggested in humans but not yet properly studied)

Question 24

is mTOR activated in feeding or in starvation?

Answer 24

mTOR is activated during feeding and inhibited when starved.

Question 25

What are some common targets between GPCR and RTK signalling?

Answer 25

PKA, PKB, PKC, CaM-kinase, MAP kinase, PLC, which themselves in turn can have common targets.

Factors from one pathway can also influence factors in another, such as Ras influence on PI3K.

Question 26

The beta-adrenergic receptors in the heart elicit much stronger responses through PKA than the alpha-receptors through IP3. True or False?

Answer 26

True.

End result of all pathways is muscle contraction through elevated Ca2+ but different secondary inputs are possible.

Question 27

How does the b1-adrenergic receptor promote cardiac muscle contraction?

Answer 27

Ligand binds to b1 receptor (adrenaline), coupled with G-alpha-s which when activated promote adenylyl cyclase to synthesise cAMP, which activates PKA, triggering a release of Ca2+ from the sarcoplasmic reticulum, triggering muscle contraction.

Question 28

How does b2 adrenergic receptor activity modulate b1 adrenergic activity in the heart?

Answer 28

b2 receptor coupled with G-alpha-i/o which when activated by ligand binding activates PDE3 which inhibits cAMP activity, reducing

Question 29

Why is the b2 adrenergic receptor needed to inhibit b1 activity in the heart?

Answer 29

Prolonged activation of b2 leads to PDE3 activation that inhibits cAMP mediated signalling. This is a protective mechanism of overexertion that can result from adrenaline activity on b1.

Question 30

How is the prolonging of adrenaline being bound to a receptor, e.g. b2 receptor, determined?

Answer 30

The prolonging is determined by the dynamics of the interactions between the ‘wobbling’ alpha-helices of the b2 adrenergic receptor and the intracellular proteins

Question 31

How does insulin activity regulate adrenaline activity on blood glucose levels?

Answer 31

Insulin activates PKB, which activates PDEs, degrading cAMP, which is generated by GCPR signalling by adrenaline.
PKB inhibits GSK, activating GS and promoting decrease in blood sugar levels through glycogen synthesis.
Adrenaline activates cAMP and therefore PKA which inhibits GS - acting on the same target in different ways.

Question 32

Briefly outline what occurs at each compartment within FGF signalling.

Answer 32

Extracellular space: FGF ligand -> Activated FGF receptor complex

Cytoplasm: Intracellular domain of complex -> activated Ras -> MAPK cascade -> activate MAPK

Nucleus: MAPK -> target transcription factors -> altered transcription of multiple target genes.

Question 33

What is the MVB?

Answer 33

Multi-vesicular Body, a structure of internal vesicles formed from invaginations of their own limiting membrane.

Question 34

What is the role of the MVB?

Answer 34

Endocytosed receptors can still be signalling due to tight ligand binding. This could be detrimental to the cell; once inside the internal vesicle of the MVB the receptor is sequestered from the cytosol preventing signalling propagation.

This is known as ‘Receptor down-regulation by sequestration’

Question 35

Where do receptors go after being transported to the MVB?

Answer 35

Can return to plasma membrane or be target of destruction factors.

Question 36

How do phosphatidylinositol phosphates (PIPs) establish compartmentalisation of signalling?

Answer 36

Phosphoinositides can be interconverted by kinases and phosphatases, allowing the specific co-localisation of signalling components and therefore their activation due to different PIPs having different target domains.

Question 37

Which compartment is PI3P localised in and what are its target domains?

Answer 37

Endosome; FYVE and PX

Question 38

Which compartment is PI4P localised in and what are its target domains?

Answer 38

Golgi; PH

Question 39

Which compartment is PI(3,4)P2 localised in and what are its target domains?

Answer 39

Multi-vesicular body; PH

Question 40

Which compartment is PI(4,5)P2 localised in and what are its target domains?

Answer 40

Plasma membrane; PH, FERM, ANTH, ENTH

Question 41

Which compartment is PI(3,4,5)P3 localised in and what are its target domains?

Answer 41

Plasma membrane; PH