Receptor Mechanism II

Question 1

The structural features of a GPCR

Answer 1

• G-protein couple or Metabotropic receptors
• 7 transmembrane domains
• 1 protein in functional GPCR
• Agonist binds in amino terminus (end bit sticking out)
• G-protein binds to C-terminus (in cytoplasm)
• The G-protein consists of a family of three proteins that bind to GDP/GTP.
• These guanosine proteins are abbreviated to G-proteins
• Trimer- all called G-proteins, even though only alpha binds.

Question 2

The mechanism that GPCRs produce a change in cellular activity.

Answer 2

• In absence
o When there is no receptor agonist, where the receptor isn’t activated, all three proteins are bound together.

• In presence of agonist
o The receptor is activated (pink circle) by the agonist binding to the protein embedded in membrane.
o At this point the receptor is bound to a g-protein (which has an alpha-subunit with GDP, covered by beta and gamma subunits)
o Upon activation, the alpha sub-unit drop the GDP and exchanges it with GTP, as it has a higher affinity for GTP than GDP.
o The alpha subunit leaves the beta and gamma subunit anchored onto membrane, and modifies and interacts with the enzyme.
o At the same time, the GTP is hydrolysed over time to GDP by the alpha-subunit and returns back to the beta and gamma sub-unit- back where it started

Question 3

Discuss how GPCRS differ in terms of downstream signals.

Answer 3

-

Question 4

Highlight the variation in signal cascade

Answer 4

In a cardiac myocyte
• 1 adrenaline molecule + 1 Beta-adrenaline receptor = 10,000 cAMP molecules released
• Activates protein kinase A and can activate and phosphorylate a number of different downstream proteins – profound effects on cellular responses.

Question 5

What enzymes are altered by activated alpha subunits?

Answer 5

• Alpha-Gs
o Stimulates adenylate cyclase
o Which makes cAMP from ATP
o If receptor couples with Gs alpha – cAMP made

• Alpha Gi
o Same receptor but couples to Gi and not Gs
o Inhibits adenylate cyclase
o Which results in reduction in cAMP

• Alpha Gq/G11
o Activates enzyme called phospholipase C
o Turned on

Question 6

Adenylate Cyclase

Answer 6

• The activated receptor likes to interact with alpha-Gs form of alpha subunit.
• As the alpha subunit has been activated, the alpha GTP stimulates adenylate cyclase
• Creates cAMP from ATP – which leads to many consequences
• cAMP can turn on activated protein kinase A, which phosphorylates many proteins
• Activated receptor -> increases cAMP -> activates protein kinase A (PKA) -> then phosphorylates many proteins.
• cAMP broken down by phosphodiesterase
• This is targeted pharmacologically
• Heart failure drugs are phospholipid inhibitors etc.

Question 7

Phospholipase c

Answer 7

• GPCR interacts with alpha Gq/G11, the alpha subunit (after leaving beta gamma anchors) interacts with phospholipase C
• Works on minor membrane phospholipid called phosphatidylinositol bisphosphate - PIP2
• Cleaves at phospholipid, creating two signal molecules
• IP3 - Inositol trisphosphate - water soluble, and goes into cytoplasm
• DAG - Diacylglycerol – stays in lipid phase
• IP3 binds to its receptor - located in intracellular calcium stores in ER lumen, and causes release of calcium and therefore an increase in calcium.
• DAG – activates various ion channels and activates protein kinase C etc.
• Any receptor coupled to alpha-Gq will increase in phospholipase C activity and an increase in calcium
• If it’s a muscle cell – get contraction (as an increase in calcium)
• If it’s a secretory cell – increase in secretion (as calcium causes secretary granules to occur

Question 8

The same acetylcholine molecules can produce very different responses. LGICR - nicotine AChR vs GPCR - muscarinic receptors

Answer 8

LGICR – nicotinic AChR
Fast Response
Contract skeletal muscle
Nicotine only activate specific LGICR
Always change in membrane potential
Rarely involved in changes in enzyme levels
5 similar protein – making an aqueous pathway

Slow Response
Slows heart rate/ increase in bowel activity
Muscarinic only activates GPCR
May not have change in membrane potentials unless downstream consequences
Always change in enzyme levels
1 proteins with G-proteins

Question 9

G alphas s

Answer 9

Adenylyl cyclase
ATP
Increases cAMP
Protein kinase A
Ion channels

Question 10

G alpha i

Answer 10

Adenylyl cyclase
ATP
Decrease cAMP
From protein kinase a = enzymes, transport proteins

Question 11

G alpha q/11

Answer 11

Phospholipase c
PIP2
IP3 or DAG
Ca2+ or protein kinase c
Contractile proteins or ion channels