Lecture 5 - G Protein Coupled Receptor Mediated Signalling

Question 1

2 key types of receptor?

Answer 1

GPCR and enzyme linked receptors.

Question 2

How many transmembrane domains do GPCRs have?

Answer 2

Seven.

Question 3

Give an example of a GPCR.

Answer 3

Beta adrenergic receptor. Ligand = adrenaline, which binds to the extracellular side between TM helices 3,5 and 6. Possesses a lipid end that anchors the C-terminus to the membrane. Couples to G proteins via the 5 and 6 TM helices.

Question 4

GPCRs couple to trimeric G proteins that consist of?

Answer 4

An alpha, beta and gamma subunit.

Question 5

There are multiple classes of trimeric G proteins, the main one being Gs, which activates adenylate cyclase. Other classes?

Answer 5

Golf = involved in smell. 
Gi = inhibits adenylate cyclase. 
Go/q = activate the PLC/Ca2+ pathway. 
Gt = transducin. Coupled to photoreceptors.

Question 6

Inactive G proteins are tightly bound to GDP. How does nucleotide exchange occur?

Answer 6

Guanine nucleotide exchange factor (GEF).

Question 7

An active G protein (GTP bound) has a low GTPase activity. How is this enhanced?

Answer 7

GTPase activating protein (GAP) is used for hydrolysis.

Question 8

Upon activation Galpha subunit can interact with target proteins on the membrane. Give an example.

Answer 8

Adenylate cyclase.

Question 9

Provide examples of some secondary messengers in signalling cascades.

Answer 9

cyclic AMP (cAMP), Ca2+, 1-2-diacylglycerol.

Question 10

What are secondary messengers?

Answer 10

Small molecules produced upon activation that usually amplify a signalling cascade. They have their own secondary messengers.

Question 11

How was cAMP discovered?

Answer 11

Liver cells were fractionated to separate the cytoplasm and the membrane. The membrane contains the GPCR, G proteins and adenylate cyclase, whilst the cytosol contains the glycogen phosphorylase.

Adrenaline added to the membrane solution and cAMP produced. cAMP then added to cytosol –> glycogen phosphorylase activated. This is proof that a small molecule transfers the signal.

Question 12

How does cAMP function?

Answer 12

It activates protein kinase A (PKA).

Question 13

Glycogen metabolism - what breaks down glycogen?

Answer 13

Glycogen phosphorylase.

Question 14

Glycogen metabolism - what synthesises glycogen?

Answer 14

Glycogen synthase.

Question 15

Effects of cAMP on glycogen degredation?

Answer 15

• Adrenaline activates G protein, binds to adenylate cyclase.
• Activates the synthesis of cAMP.
• cAMP activates PKA, which phosphorylates glycogen phosphorylase kinase (GPK).
• Glycogen phosphorylase is phosphorylated by GPK and glycogen breakdown is activated.

ALSO:

• PKA phosphorylates protein phosphatase (PP) = inactivated.
• PP is responsible for the removal of the phosphate from glycogen phosphorylase and GPK, inactivating them both.
• PKA phosphorylates glycogen synthase = inactivated.

Question 16

The role of PKA and PP in glycogen synthesis?

Answer 16

cAMP does not activate PKA. Since PKA is inactive, PP is active. PP removes the phosphate from glycogen phosphorylase and GPK, inactivating them both.

PP will dephosphorylate glycogen synthase = activated.

Question 17

How do cells maintain a low level of calcium in their cytosol, compared to outside the cell/in the ER?

Answer 17

Difference maintained by calcium ATPases –> pump calcium out of the cell/into organelles.

Question 18

Calcium channels on the p. membrane/ER when activated provide an increase in cytosolic Ca2+. Occurs when required as a secondary messenger. Name the channels on each membrane.

Answer 18

Plasma membrane channels: CRAC and P2X.

ER channels: IP3R. ER channels have a clacium pump, SERCA, that constantly removes excess cytosolic calcium.

Question 19

Examples of calcium signalling?

Answer 19

Muscle contraction and NT release.