Lecture 5 - GPCRs & G-proteins

Question 1

What is another name for GPCRs (G-protein coupled receptors)?

Answer 1

Metabotropic receptors

Question 2

Do GPCRs have ion channels?

Answer 2

no - however G-proteins can be activated

Question 3

What type of binding site do GPCRs have?

Answer 3

Neurotransmitter binding site

Question 4

What 3 G-subunits are attached to GPCRs?

Answer 4

Alpha, beta & gamma G-proteins

Question 5

What leads to the G-protein (inactive trimer) being activated?

Answer 5

GDP is bound till activation following binding of GTP. This leads to a signalling cascade.

Question 6

What are G-protein coupled receptors?

Answer 6

Cells use approximately 25 different families of receptor proteins to detect and respond to a myriad of chemical and physical stimuli - GPCRs are the most diverse.

Question 7

What are the structural features of GPCRs?

Answer 7

• 7 transmembrane (a helices) of all GPCRs are packed in a similar way
• TM3 centrally located next to binding pocket, crucial for ‘transduction’ of ligand binding
• other transmembranes and extracellular N+ terminus also contribute to ligand binding
• N terminal on extracellular side and where the ligand binds. C terminal where G-protein is bound on the intracellular side

Question 8

What terminal is the G-protein bound to?

Answer 8

C terminal

Question 9

What determines GPCR classes?

Answer 9

• structural features of the extracellular domains defining the ‘ligand’ binding site
• linked directly to the huge diversity of the stimuli GPCRs can detect.

Question 10

What are different types of ligands?

Answer 10

Amino acids, hormones, proteins etc.

Question 11

What determines what ligand will bind?

Answer 11

Differences in the binding pocket. Different cell types express different amounts and types of receptors

Question 12

What is an example of a protease-activated receptors (PAR) in platelets?

Answer 12

Receptor activated by cleavage of the N terminal which in turn acts as a tethered ligand - part of the receptor itself acts as the agonist.
- Receptors work together to elicit a response - 3 independent stimuli activate platelets
- N-terminal acts as its own ligand, as it is cleaved by thrombin
- The resting platelet expresses 3 types of receptors - binding of collagen receptors leads to activation of ADP, leading to that receptor becoming activated. Meanwhile the thrombin receptors are activated

Question 13

What 3 receptors are found on platelets?

Answer 13

• collagen receptors
• ADP receptors
• thrombin receptors

Question 14

Which side of the GPCR is the N & C terminus found?

Answer 14

N-terminus at extracellular - ligand binding
C terminus at intracellular - G-protein binding

Question 15

What are G-proteins?

Answer 15

• Guanine nucleotide-binding proteins - belong to the GTPase family
• Act as molecular switches inside cell to transmit signals from extracellular stimuli
• Regulated by ability to bind and hydrolyse GTP (‘on’) to GDP (‘off’)
• Exist as heterotrimeric complexes made up of a, B and y subunits

Question 16

What occurs when the GDP is bound to the alpha subunit?

Answer 16

The G-protein is in an inactive state

Question 17

How is G-protein signalling controlled?

Answer 17

• G-proteins are timers
• Duration of signalling by activated trimeric G protein is regulated by rate of GDP hydrolysis by Ga(alpha)
• RGS (regulators of G-protein signalling) proteins stimulate GTPase activity in the a subunit

Question 18

What is RGS?

Answer 18

Regulators of G-protein signalling

Question 19

How many different families of G-proteins exist?

Answer 19

6:
Gs
Gi
Gq
G12/13
Gti (Transducin)
Gz

Question 20

What does Gs do?

Answer 20

Stimulates adenylyl cyclase (increase cAMP)

Question 21

What does Gi do?

Answer 21

Inhibits adenylyl cyclase (decrease cAMP) and activates phospholipase CB

Question 22

What does Gq do?

Answer 22

Activates phospholipase CB, increasing IP3, DAG and intracellular calcium

Question 23

What does G12/13?

Answer 23

Modulates Rho GTPases, affecting the cytoskeleton and cell migration

Question 24

What does Gti (Transducin) do?

Answer 24

Involved in visual signaling through rhodopsin and phosphodiesterase activation

Question 25

What does Gz do?

Answer 25

Regulates ion channels and modulates neurotransmitter release and platelet aggregation

Question 26

What does G(olf)a subunit do?

Answer 26

Subunit & cell type specificity to detect smell

Question 27

How do organisms respond specifically to particular stimuli?

Answer 27

Selective expression of certain receptors and the molecules involved in signal transduction allow cells to respond specifically to particular stimuli

The cell type and where it is can also create diversity.

It is the specific subunit and cell type that determine the response using the same signalling as the other cell type

Question 28

Describe how effectors are determined by the class of Ga (alpha) subunit

Answer 28

Effectors include enzymes that create 2nd messengers and ion channels whose gating is regulated either directly (By subunits), or indirectly by 2nd messengers and their effectors

Question 29

Describe the process of direct activation of an ion channel

Answer 29

• Similar mechanism as ligand-gated channels
• Slow to open or close
• Stay open or closed for longer - minutes rather than milliseconds

Question 30

Describe how activated G proteins regulate the activities of enzymes that control the levels of second messengers

Answer 30

Second messengers are small molecules that carry signals inside the cells

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 31

Why do we need these second messenger system?

Answer 31

A single ligand binding to a single GPCR results in the phosphorylation (activation) of millions of proteins.

Question 32

Describe how cholera toxin leads to disease?

Answer 32

Vibrio Cholera - Gs a subunit (stimulatory). The second messenger activates adenylate cyclase = catalyses cAMP = activation protein kinases. The effect on the cell is an increase in Cl- ion secretion = increase in Na+ ion secretion + H2O. The physiological effect is excess fluid and electrolytes in the lumen of the small intestine, leading to Diarrhoea & extreme dehydration.

Question 33

Describe how a whooping cough leads to disease?

Answer 33

Bordatella pertussis - Gi a subunit (inhibitory). Secondary messenger inactivates inhibitory G protein = increase in cAMP. This leads to too much intracellular cAMP. The physiological effect is that it leads to erosion of the respiratory epithelium and large quantities of mucus-containing fluid. This triggers coughing fits

Question 34

What is an example of mutations in GPCRs?

Answer 34

Uveal melanoma - GNAQ and GNA11
- over 90% of uveal melanoma have mutations in Gq (a) subunit
- leads to blocking of GTP hydrolysis so subunits always active causing permanent signal transmission (constitutively active growth pathways)
- thought to occur early on in tumour development
- not yet targeted for therapy but can be used for diagnosis