Lecture 6 cont (revise L7 before this)

Question 1

What are the 3 “superfamilies” of cell surface receptors?

Answer 1

1) GPCR’s
2) Ligand gated ion channels (ionotropic receptors)
3) Receptors with intrinsic enzyme activity e.g.: tyrosine kinase receptors (covered in lecture 6, part 1)

Question 2

Describe the difference between ligand agonists and antagonists.

Answer 2

Agonists = bind to produce a biological effects
Antagonists = bind but dont activate, simply block the effects of the agonists.

Question 3

What is the common basic structure of a GPCR?

Answer 3

• 7 transmembrane domains (7TMD) as a single polypeptide chain (300-1200 AA’s). These are folded to form a binding domain of the agonist.
• with an extracellular N-terminus and an intracellular C-terminus.
• Different G proteins, have different subunits, that do different things (lecture 7)

Question 4

Structure of GPCR:

• Name of the domains
• Where are the 2 regions of binding for ligands in GPCR’s?

Answer 4

Binding domains:

1) In between TM domains 2&3 (most common)
2) Extracellular domains within the N-terminus

• G-protein coupling domain

Binding of the agonist, changes the conformation of the G protein coupling domain

Question 5

What occurs in the GPCR after ligand binding?

Answer 5

• Conformational change in G-protein receptor
• Activation of G-protein, GDP on alpha subunit switched for GTP
• Dissociation of alpha and beta-gamma subunits, which interact with effector proteins

Question 6

Talk through what happens with G-protein coupling with Gs…

Answer 6

• At rest, the receptors are coupled to a G protein (G protein is a heterotriimeric protein of Gs alpha with beta and y subunit. The GsAlpha subunit is bound to GDP
• Binding of adrenaline causes the GDP to dissociate and is replaced by a GTP. The binding of adrenaline caused a conformational change of Gsalpha which caused it to lose it’s affinity for GDP and GTP to replace the GDP
• This results in the disociation of subunits from the receptor, producing Gsalpha with GTP complex and a beta-gamma complex
• Both complexes go on to activate an effector
• For example, Gsalpha goes on to activate an enzyme called adenyl cyclase which converts ATP to cyclic AMP. cAMP is a second messanger, that goes on to take the message into the cell

Question 7

How are G-protein signals terminated?

Answer 7

• GTPase hydrolyses GTP on alpha subunit back to GDP
• affinity of alpha subunit for beta-gamma subunit increases, they reform and go back into resting state

Question 8

What secondary messenger does Gs, Gi and Gq initially activate?

Answer 8

Gs = Adenylyl cyclase (stimulates)
Gi = Adenylyl cyclase (inhibits)
Gq = Phospholipase C (stimulates)

Question 9

Amplification in cellular signalling - G proteins

Answer 9

• An activated receptor will activate 4 G proteins on average
• Each activated effector is a proteins that converts ATP to cAMP. Lots of ATP are converted per effector
• cAMP then goes on to activate protein kinase A
• Protein kinase A goes on to phosphorylate thousands of substrates (e.g. enzyme X). Each protein kinase will activate a similar number of enzyme X.
• Enzyme X may then go on to phosphorylate another thousand molecules

FROM ONE BINDING EVENT AT THE BEGINNING, CAN LEAD TO HUGE AMPLIFICATION

Question 10

How does pertussis (whooping cough) caused by Bordetella pertussis interfere with G-protein signalling?

Answer 10

Covalently modifies G-protein such that GTP cannot be exchanged for GDP which means the G-protein cannot be activated. - It uncouples Gi-preferring GPCR’s from mediating signal transduction events.

Question 11

How does Cholera, caused by vibrio cholerae interfere with G-protein signalling.

Answer 11

Prevents termination of signalling by Gs-preferring GPC’s leading to long lasting activation of downstream pathways.

Question 12

4th superfamily: Intracellular receptors (structure)

Answer 12

Question 13

Intracellular receptor - how does it work?

Answer 13

Can see in the top of the pic, the DNA binding site bound inhibitory protein complex, currently at rest so inactive as inhibitory protein complex is bound

• The receptor is bound to an inhibitory protein complex
• At rest, the receptor is prevented from binding to DNA by the binding of the inhibitory protein complex on it’s DNA binding site
• Binding of the hormone to the hormone binding site. The inhibitor undergoes a large conformational change. It then disociates the inhibitory protein complex and revels the DNA binding sites. This DNA binding site can go on to bind it’s binding site in DNA and then affect transcription

Question 14

What is the other name of intracellular receptor?

Answer 14

Nuclear receptor