Lecture 10 - GPCRs

Question 1

Describe the basic structure of GPCRs

Answer 1

Structure
* 7 transmembrane α-helical regions
* The G proteins interact with α-helices 5 and 6
* There are 4 cytosolic domains and 4 extracellular domains
* There is an 8th smaller α-helix which runs parallel to the face of the membrane

The Principle groups of G protein-coupled receptor

Family A - Basic structure (Biological amines, Light or odorants, Peptides or chemokines, purines, lipids )
Family B - Have a large extracellular domains associated with the perception of stimuli (Peptides)
Family C - Frequently form dimers, they have a large Venus fly trap domain attached to the N-terminus. Reception occurs between the jaws (in pocket) and is sensed by the interactions of two parts of the domain. (Biological amines, Glutamate, Ca2+)

Question 2

How does Epinephrine cause a responce through the B-adrenergic receptor?

Answer 2

The fight or flight hormone and a type of catecholamine released by the adrenal medulla. Increases glycogen breakdown and release of fatty acids.

1. Epinephrine interacts with 15 amino acids (Mainly serine and asparagine) on four of the 
transmembrane α-helices (3, 5, 6 and 7).

2. When the epinephrine binds to the  β-adrenergic receptor it interacts with G-proteins specifically a type called GS proteins and activates it. 
Heterotrimeric G-proteins have subunits. (α, β, γ)

3. The G protein is activated by the hormone binding to the protein coupled G-protein receptor. The GDP bound in the guanyl nucleotide binding site is exchanged for GTP and the βγ subunits are lost which can also stimulate downstream signalling. (later on it can be deactivated by the Hydrolysis of GTP to GDP through intrinsic GTPase activity of the α subunit and the βγ re-binds)

4. The α unit diffuses to the target (adenylate cyclase) cyclase has 2 regions each with 6 TMS domains. On the cytosol side it has two catalytic domains which turn ATP into Cyclic AMP which activates protein kinase A. 

Kinase A increases lipid breakdown and decreases glycogen synthesis.

Question 3

How is protein Kinase A activated

Also discuss signal termination and regulation of activators.

Answer 3

Protein Kinase A - cAMP dependent kinase
When unstimulated the pseudo substrate sequence which occludes the active site of the catalytic domains. When 4 cAMP binds to the regulatory substrates and the regulatory domains move away and the pseudo substrate sequence is removed from the active site activating it.

The signal is terminated by:
* Dissociation
* Phosphorylation by GPCR kinase (phosphorylation of region normally interacting with g protein) and binding of arrestin
Endocytosis

Regulation of cAMP levels
The adenylyl cyclase activity can either be activated or inhibited through either inhibatory or stimulatory hormones binding to receptors.

Question 4

what is the signalling pathway associated with a-adrenergic receptor?

Answer 4

1. The ligand binds to the α1-adrenergic receptor
   
   2. This activates the G protein
   3. The α - subunit activates phospholipase C
   4. This causes PIP2 to cleave into IP3 and DAG
   5. DAG is a lipid so diffuses in the membrane
   6. The IP3 migrates to an IP3 receptor in the ER membrane
   7. This causes the release of calcium
   8. The Ca2+ and DAG activates protein kinase C

Activation of protein kinase C occurs as the binding of DAG causes a conformation shape change which allows calcium ions to bind, which causes a conformational shape change removing the pseudo substrate from, binding at the active site.