Lecture 6 - GPCRs II (signaling)

Question 1

What are effectors of trimeric G proteins?

Answer 1

Enzymes that create messengers and ion channels whose gating is regulated either directly (by subunits) or indirectly by 2nd messengers and their effectors

Question 2

What are secondary messengers?

Answer 2

small molecules that carry signals inside cells

Question 3

What activities are regulated by activated G protein?

Answer 3

the activities of enzymes that control the levels of second messengers. These include:
- Hydrophobic lipids confined to the membrane in which they are generated
- Small soluble molecules that diffuse through the cytoplasm (cAMP, cGMP)
- calcium ions

Question 4

Describe features of the cAMP second messenger system - Adenylate cyclase

Answer 4

• 10 isoforms
• membrane anchored enzyme
• activated by Gas, inhibited by Gai

Question 5

What are the steps in cAMP second messenger system?

Answer 5

1. Ligand binds to receptor activating G protein
2. A subunit moves and binds to adenylate cyclase in the membrane
3. This activated enzyme catalyse formation of a cAMP from ATP
4. The cAMP (2nd messenger) activates Protein Kinase A (PKA)
5. PKA phosphorylates/activates protein
6. Initiates a response within the cell

Question 6

Explain the process of B2 adrenoreceptor regulation of metabolism in liver and skeletal muscle

Answer 6

Binding of single epinephrine molecule to a receptor sets off a signalling cascade, resulting in phosphorylation/activation of enzymes controlling glycogen metabolism

Signal is switched off by:
- agonist dissociating from receptor
- GTPase activity of Gas
- cAMP breakdown by phosphodiesterase
- Dephosphorylation of enzymes

Question 7

What are the differences in the cGMP second messenger system and cAMP?

Answer 7

cGMP:
- enzyme guanylate cyclase which can be receptor bound or ‘free’ in the cytoplasm
- converts guanosine triphosphate (GTP) to 3’,5’-cyclic guanosine monophosphate (cGMP)

Question 8

Describe how the message is encoded in the concentration & frequency of changes in concentration

Answer 8

Local concentrations of 2nd messengers are determined by rate of production, rate of diffusion from site of production and rate of removal.

Question 9

What is cAMP production regulated by?

Answer 9

Adenylyl cyclase

Question 10

What is involved in the removal of cAMP?

Answer 10

phosphodiesterase

Question 11

What may membrane lipids be targeted by?

Answer 11

Receptor regulated lipases, to generate 2 kinds of 2nd messengers:
- (i) will be water soluble & diffuse through the cytoplasm - e.g. IP3
- (ii) hydrophobic molecules, which will remain in the membrane - e.g. DAG

Question 12

What do lipase kinases do?

Answer 12

Add phosphate groups to lipids - e.g. DAG to male phosphatidic acid, or to PI to generate PIP, PIP2, PIP3

Question 13

What can GPCRs activate?

Answer 13

PLC (Phospholipase C) to generate IP3 & DAG

Question 14

What does specificity in signalling come from?

Answer 14

Comes from selective expression and cellular localisation of signalling molecules
- e.g. numerous isoforms of PLC and PKC are found in cells
- PKCs (Protein Kinase C) are Ser/Thr kinases, activated by DAG (C1 domain) and Ca2+ (C2 domain)
- PMA (Phorbol Myristate Acetate) is an analogue of DAG used in research to activate PKCs.

Question 15

What are targets of phospholipase CB (PLC)?

Answer 15

• activation of protein kinase C (PKC)
• DAG binding causes dissociation of intramolecular pseudosubstrate domain from active site, once activated PKCs can provide either positive or negative feedback in signalling pathway

Question 16

What does phosphorylation of PLCB lead to?

Answer 16

It provides negative feedback for GPCR signalling, makes the signalling ‘transient’.

Phosphorylation of receptors can contribute to desensitization

Question 17

What is the role of phospholipase CB?

Answer 17

activation of Protein Kinase C (PKC)

Question 18

What does Calcium do?

Answer 18

Regulates diverse cell functions

Question 19

What processes are regulated by Calcium signalling?

Answer 19

• synaptic transmission
• hormone secretion & synthesis in some cases
• fertilisation
• muscle contraction
• cytokinesis

In resting cells, cytosolic (Ca2+) is kept low (approx. 100nm) by ATP-driven Ca2+ pumps. This is important for it to be able to act as a secondary messenger.
- receptors regulate the activity of Ca2+ channels to produce transient rises in Ca2+

Question 20

How is calcium regulated as a second messenger?

Answer 20

1. Calcium influx into the cytosol is regulated by channels in the extracellular membrane AND ligand-gated channels on the ER.
2. Store-operated channel made up of ORAI and gated by STIM are responsible for store refilling and maintaining ER calcium levels.
3. Important role in activation of T-lymphocytes - loss of function mutation in Orai1 causes sever combined immunodeficiency (SCID)

Question 21

Describe why Ca imaging with fluorescent tags is useful

Answer 21

• used to study signalling in a wide variety of cells
• in response to many types of stimuli
• to gain insight into disease mechanisms and develop new drugs

Question 22

Describe the process of de-sensitization of GPCRs

Answer 22

Overstimulation of GPCRs =
- Tachyphylaxis - e.g. LSD or salbutamol
- Disease - e.g. uncontrolled growth in cancer

2 mechanisms - GRK (G-protein coupled receptor kinases) and B-arrestin

1) stops G-protein from binding
2) internalises receptor - degraded or recycled

Question 23

Describe the implications of desensitisation of GPCRs for medicine

Answer 23

• prolonged photon response and rod apoptosis
• embryonic lethality