M&R 7.2 G-protein effector mechanisms

Question 1

What does the Gs alpha subunit do?

Answer 1

Stimulates adenylyl cyclase, which converts cellular ATP –> cAMP
cAMP interacts with Protein Kinase A (PKA) to activate it
PKA can phosphorylate other proteins in the cell to regulate their activities

Question 2

How does increased cAMP activate Protein Kinase A?

Answer 2

PKA has both regulatory (R) and catalytic (C) subunits
When R subunits are bound to C subunits, the C subunits are not capable of their activity
Increased cAMP binds to R subunits and causes them to dissociate from C subunits - therefore C subunits are activated

Question 3

Once activated, how does Protein Kinase A exert its effects?

Answer 3

The C (catalytic) subunits phosphorylate serine and threonine residues on target proteins in the cell.

This can alter the activity of a protein, its location within the cell, or make it more susceptible to degradation.

Question 4

What does the Gi alpha subunit do?

Answer 4

Inhibits adenylyl cyclase

therefore cAMP is not generated from ATP and PKA does not get activated

Question 5

What does the Gq alpha subunit do?

Answer 5

Activates phospholipase C (PLC)

PLC cleaves PIP2 (an amphipathic membrane phospholipid) into IP3 (polar part) and DAG (lipid part)

Question 6

After IP3 is produced from PIP2 by PLC, what does it do?

Answer 6

It is polar so can travel in the cytoplasm
It travels to the ER and interacts with ligand-gated ion channels (IP3 receptors) so that Ca2+ is released from the ER into the cytoplasm
(can therefore lead to activation of Ca2+-dependent protein kinases, e.g. Ca2+/calmodulin-dependent protein kinase [CaM-kinase])

Question 7

After DAG is produced from PIP2 by PLC, what does it do?

Answer 7

DAG is hydrophobic so it stays in the membrane
It interacts with Protein Kinase C (PKC) in the membrane to activate it
PKC can phosphorylate other proteins to change their activity

Question 8

What does the Gt alpha subunit do?

Answer 8

Rhodopsin GPCR activated by light
Gt (transducin) activates cGMP phosphodiesterase
cGMP PDE breaks down second messenger (cGMP) to 5’GTP

So in dark there is lots of cGMP but in light there is less - this affects ion channels and therefore MP - alters signal to CNS

Question 9

How do the actions of protein kinases differ from each other?

Answer 9

Each protein kinase causes phosphorylation (of serine/threonine residues) of a distinct family of target proteins

(e.g. enzymes, ion channels, structural proteins etc)

Question 10

What is an important role of receptor->G-protein->effector signalling systems?

Answer 10

Amplification of the signal

Question 11

Describe some levels at which signal amplification can occur in receptor-Gprotein-effector mechanisms

Answer 11

1. An activated receptor can sequentially activate >1 G-protein
2. An activated alpha-GTP or beta-gamma subunit can activate multiple effector molecules
3. Effector molecules act catalytically (activation of an enzyme converts thousands of molecules, opening of an ion channel moves thousands of ions)

Question 12

What factors promote deactivation of GPCR signalling pathways at the level of receptors?

Answer 12

1. agonist-receptor binding is weakened after the receptor has interacted with the G-protein - makes dissociation of agonist more likely
2. while activated, the receptor is susceptible to protein kinases which phosphorylate it to prevent it activating further G proteins

Question 13

What factor promotes deactivation of GPCR signalling pathways at the level of the G-protein?

Answer 13

Cellular factors can stimulate the intrinsic GTPase activity of the alpha subunit, therefore reducing the lifetime of the alpha-GTP subunit

Question 14

What factors promote deactivation of GPCR signalling pathways at the level of enzymes?

Answer 14

1. There are lots of enzymes in the cell that metabolise 2nd messengers to return the cell to basal levels (e.g. phosphodiesterases (PDE) convert cAMP –> 5’AMP (non-active))
2. Enzyme cascades downstream of 2nd messenger/PK activation are opposed by activities which reverse the effects (e.g. target protein phosphorylation opposed by cellular protein phosphatases)

Question 15

Explain the mechanism by which adrenaline (in blood) and noradrenaline (released from SNS) act on beta1 adrenoceptors in the ventricle to increase force of contraction (positive inotropy)

Answer 15

Beta1 receptors have Gs subunit -> activates adenylyl cyclase to increase cAMP -> cAMP activates PKA
PKA phosphorylates L-type Ca2+ channels so more Ca2+ can enter per depolarisation
This triggers CICR via ryanodine receptors -> increased [Ca2+]i -> increased contractility via excitation-contraction coupling

Question 16

Explain the mechanism in smooth muscle by which NA from the SNS acts on alpha1 receptors to produce vasoconstriction, and also ACh from the SNS acts on M3 receptors to produce bronchoconstriction

Answer 16

Both alpha1 & M3 use Gq subunit
Gq activates PLC to produce IP3 (increases Ca2+ release from ER) and DAG (activates PKC)

Increased [Ca2+] and PKC activity leads to increased contractility and therefore constriction

Question 17

Explain the mechanism by which neurotransmitter release in the CNS & PNS can be modulated by pre-synaptic GPCRs on the nerve terminal

Answer 17

The beta-gamma subunit can inhibit certain types of voltage-gated Ca2+ channels
Therefore makes it more difficult for Ca2_+ to enter the nerve terminal on each depolarisation
reduced Ca2+ influx leads to reduced NT release