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Flashcards in Session 7 Deck (21):

Describe the structure of G proteins

Heterotrimeric - three distinct subunits - alpha, beta and gamma. Beta and gamma bind tightly together and function as a single unit. The alpha subunit has a guanine nucleotide binding site which binds to GTP and slowly hydrolyses it to GDP.


What happens to the G protein before and after the receptor is activated?

Under basal conditions the G protein is present in heterotrimeric form with GDP bound to the alpha subunit.
The activated receptor has a high affinity for this form of the G protein and an interaction occurs, causing GTP to bind in the place of GDP. The receptor acts as a guanine nucleotide exchange factor (GEF).
The alpha-GTP and beta-gamma subunits are then released separately to interact with receptors.


How is the effector interaction of G protein subunits terminated?

By the intrinsic GTPase activity of the alpha subunit hydrolysing GTP to GDP. The affinity of the G-alpha for the G-beta-gamma subunits increases and G protein heterotrimer is reformed ready for activation.


What is the on/off switch and timer of G proteins?

On Switch - receptor mediated GDP/GTP exchange
Timer/off switch - the length of time taken for GTP hydrolysis on the G-alpha subunit


Give the mechanism of action of the Gs G protein

Catecholamines act on B adrenoreceptors with the Gs G protein to increase the activity of adenyl cyclase. This increases the conversion of ATP to cAMP, a second messenger that activates PKA to phosphorylate proteins and hence regulates them.
The G protein Gi found on M2 receptors opposes this action (signal=ACh)


Outline the mechanism of action of the Gq G protein

Acetylcholine acts on M2/M3 or Catecholamines act on A1 receptors all containing Gq G protein to stimulate phospholipase C. This increase the conversion of PIP2 (a phospholipid) to IP3 and DAG (second messengers).


Describe the mechanism of action on the Gt G protein

Light activated rhodopsin, which stimulates cGMP phosphodiesterase, increasing the conversion of cGMP to 5'GMP. This breaks down the second messenger, leading to Na+ and Ca2+ channel closure and hyperpolarisation of the membrane.


Link adrenergic and cholinergic receptors to the G proteins the utilise

A1 - Gq
A2 - Gi
B1 - Gs
B2 - Gs

M1 - Gq
M2 - Gi
M3 - Gq


How does cholera toxin manipulate the G protein cycle?

It contains an ADP-ribosyltransferase that eliminates GTPase activity of Gs-alpha, causing it to be irreversibly activated.


How does the pertussis toxin (whooping cough) manipulate the G protein cycle?

It contains an ADP-ribosyltransferase that interferes with the GDP/GTP exchange on Gi-alpha leading to irreversible activation.


What forms the ligand binding site of GPCRs?

Small Ligands - 2 to 3 of the transmembrane domains within the membrane
Large Ligands - extracellular N terminal


What effects can receptors that activate adenylate cyclase have?

Increased glycolysis and Gluconeogenesis in the liver, increased lipolysis in adipose, relaxation of smooth muscle and positive ionotropic and chronotropic effects of the heart.


What effects does IP3 and DAG have in the cell?

IP3 interacts with specific intracellular receptors on the ER allowing Ca2+ to enter the cytoplasm and activate Ca2+ sensitive protein kinases.
DAG activates protein kinase C's.


List different second messengers and the protein kinase molecules they activate

cAMP - cAMP dependent protein kinase (PKA)
cGMP - cGMP dependent protein kinase (PKG)
DAG - protein kinase C
Ca2+ - Ca2+/calmodulin dependent protein kinase


On what levels can amplification of a signal occur?

Activated receptor can cause GDP/GTP binding exchange on >1 G protein
An activated G-alpha/free G-beta-gamma can activate multiple effector molecules
Effector molecules act catalytically to produce many second messengers
Second messengers may activate their cellular targets using an enzyme or enzyme cascade.


Discuss the deactivation of G protein coupled receptor pathways

Once a receptor has productively interacted with a G protein the binding of the agonist molecule is weakened and receptor-agonist deactivation is more likely to occur.
Whilst activated the receptor is susceptible to a variety of protein kinases which phosphorylates the receptor preventing it from activating further G proteins
The lifetime of a GTP may be limited by cellular factors that stimulate intrinsic GTPase activity of the alpha subunit.
Cells contain high activity enzymes that metabolise second messengers
Enzymatic cascades are opposed


How does activation of M2 muscarinic receptors in the SA node cause an effect?

They increase the K+ permeability causing hyperpolarisation, slowing the intrinsic firing rate therefore having a negative chronotropic effect.


How does activation of B1 adrenoreceptors in the cardiac ventricles cause an effect?

Increases the open probability of VOCCs by activation directly with Gs- alpha and indirectly by cAMP. This results in a positive ionotropic effect.


How does the activation of A1 adrenoreceptors in arteries produce a response?

IP3 production via Gq is stimulated which releases ER Ca2+ and initiates a contractile response.
Activation of PKC by DAG is important for sustaining contraction.


What causes bronchoconstriction?

ACh acting on M3 muscarinic receptors


Give an example of how can pre synaptic GPCRs modify neurotransmitter release?

m-opioid receptors can be stimulated (by endogenous opioids or analgesics such as morphine) to produce Gi-alpha proteins. The G-beta-gamma subunits interact with VOCCs to reduce the entry on Ca2+, inhibiting neurotransmitter release.