Session 7

Question 0

What are the cellular targets for activated G proteins

Answer 0

Adrenaline –> b-adrenoceptor –> stimulates adenylyl cyclase (Gs)
Acetylcholine –> M2 muscarinic –> inhibits adenylyl cyclase (Gi)
Acetylcholine –> M3 muscarinic –> stimulates phospholipase C (Gq)
Light –> rhodopsin –> stimulates cyclic GMP, phosphodiesterase (Gt)

Question 1

Discuss G-proteins are their mechanism of action

Answer 1

G proteins are heterotrimeric - they are made up of three distinct subunits (a,B,y). The B and y bind tightly and function as a single unit. The a unit has a guanine nucleotide binding site which binds GTP and slowly hydrolysis it to GDP.
The G protein is present at the inner face of the plasma membrane.
Agonist binds receptor –> protein-protein interaction releases GDP, binds GTP –> aGTP and By released and interact with effectors –> GTP hydrolysed to GDP –> aGDP and By reform heterotrimer
The on switch is receptor facilitated GDP/GTP exchange and the timer/off switch is governed by the length of time for GTP hydrolysis

Question 2

Explain the use of cholera and pertussis toxins in experimental manipulation of the G protein cycle

Answer 2

Cholera toxin and pertussis toxin are enzymes that ADP-ribosylate specific G proteins.
CTx eliminates the GTPase activity of Gsa - this leads to Gsa becoming irreversibly activated
PTx interferes with the GDP/ GTP exchange on Gia - this leads to Gia becoming irreversibly inactivated

Question 3

Describe some diseases caused by mutations to GPCRs

Answer 3

Retinitis pigmentosa - loss of function mutation to rhodopsin
Nephrogenic diabetes insipidus - loss of function mutation to V2 vasopressin receptor
Familial male precocious puberty - gain of function mutation to LH receptor

Question 4

Summarise the second messenger generating effectors for GPCRs adenylyl cyclase, phospholipase C, cyclic GMP, phosphodiesterase

Answer 4

Gs stimulates, Gi inhibits adenylyl cyclase which hydrolysis cellular ATP to generate cyclic AMP. Cyclic AMP interacts with protein kinase A which phosphorylates other proteins within the cell.
Gq activates phospholipase C which hydrolyses PIP2 –> IP3. IP3 interacts with specific intracellular receptors on the ER to allow calcium to leave the lumen and enter the cytoplasm
Gt regulates the breakdown of the second messenger cyclic GMP phosphodiesterase

Question 5

Discuss the deactivation of GPCR pathways

Answer 5

Binding of the agonist is weakened and agonist-receptor dissociation is more likely to occur.
Whilst activated, the receptor is susceptible to a variety of protein kinases that phosphorylate the receptor and prevent it further activating G proteins (receptor desensitisation).
Basal state of enzymatic activities in the cell are favoured.
Enzymatic cascades activated downstream of second messenger activation act to oppose their effect.

Question 6

Give some examples of signalling pathways in action

Answer 6

Chronotrophy of heart - parasympathetic ACh –> M2 muscarinic –> increases open probability of K+ channels via Gi –> hyperpolarisation –> negative chronotrophy
Inotrophy of heart - sympathetic adrenaline –> b-adrenoceptors –> increases open probability of VOCC via Gs –> influx of Ca2+ –> positive inotrophy
Arteriolar vasoconstriction - sympathetic noradrenaline –> a1-adrenoceptors –> stimulate phospholipase C via Gq –> IP3 releases Ca2+ from ER –> initiates contractile response
Neurotransmitter release - presynaptic u-opioid stimulated by endogenous epioid or morphine via Gai –> GBy interacts with VOCC –> reduce Ca2+ release –> inhibits release of neurotransmitter from presynaptic terminal