GPCRs

Question 1

How many GPCRs are encoded in the human genome?

Answer 1

> 800

Question 2

Describe the common basic structure of GPCRs.

Answer 2

• Single polypeptide chain
• 7 transmembrane-spanning regions
• Extracellular N-terminal
• Intracellular C-terminal

Question 3

What is the mode of action of GPCRs?

Answer 3

1. Ligand binds to GPCR - causes R conformational change.
2. Activated GPCR facilitates GTP for GDP exchange on G alpha subunit of a G protein.
3. G protein alpha-beta/gamma complex immediately dissociates (into alpha-GTP + free beta/gamma).
4. Each subunit interacts with effector proteins:
   
   • second messenger generating enzymes
   • ion channels

Question 4

What are G proteins?

Answer 4

• Guanine-nucleotide binding protein

- Heterotrimeric: alpha, beta and gamma subunits

Question 5

What determines the duration of the alpha-GTP and/or beta-gamma interaction with effectors?

Answer 5

• G alpha GTPase activity hydrolyses GTP back to GDP - G protein subunits reform an inactive complex.
• GTPase activity is poor - up to 5 sec for hydrolysis to occur, allows time for signal transduction.

Question 6

What would be the consequence of a mutation in G alpha GTPase?

Answer 6

• Mutation that increases activity would decrease response - less time.
• Mutation that decreases activity would increase response - more time.

Question 7

What are the 3 different forms of G protein alpha subunits?

Answer 7

1. Gs - activates adenylyl cyclase
2. Gi - inhibits adenylyl cyclase
3. Gq - activates phospholipase C

Question 8

Which second messengers do Gs and Gq activate?

Answer 8

• Gs: catalyses formation of cAMP from ATP by adenylate cyclase.
• Gq: catalyses formation of IP3 and DAG from PIP2 by phospholipase C.

Question 9

Which GPCRs are associated with the activities of Gs, Gi and Gq?

Answer 9

• Alpha1 adrenoR - Gq
• Alpha2 adrenoR - Gi
• Beta1 adrenoR - Gs
• Beta2 adrenoR - Gs

Question 10

Which type of G alpha subunits are associated with cholinergic GPCRs?

Answer 10

• M1 & M3 (muscarinic R): Gq - stimulates phospholipase C
• M2: Gi - inhibits adenylyl cyclase

Question 11

How do the cholera and pertussis toxins interfere with G protein function?

Answer 11

• PTx catalyses ADP-ribosylation of Gi - prevents GPCR-G interaction.
• CTx modifies all Gs containing proteins - unable to hydrolyse GTP - activated for 18-24 hrs before being degraded - very high cyclic AMP concs - cell damage.

Question 12

What type of effectors are stimulated by GPCR and G protein activation?

Answer 12

1. Enzymes, e.g.
   
   • adenylyl cyclase (ATP to cAMP)
   • phospholipase C (PIP2 to IP3 + DAG)
2. Ion channels, e.g.
   
   • voltage-operated calcium channels
   • G protein-regulated inwardly-rectifying K+ channels (KIRKs)

Question 13

Describe the diversity of G proteins.

Answer 13

Human genome encodes:

• 20 G alpha
• 5 G beta
• 12+ G gamma

Question 14

Describe the chain of events leading to and following from Gs activation.

Answer 14

1. Agonist binds to GPCR - conformational change.
2. GPCR mediates GDP replacement by GTP on alpha subunit.
3. Alpha-GTP dissociates from beta/gamma - activates adenylyl cyclase (AC).
4. AC converts ATP to cyclic AMP (+2 Pi) - hydrophilic molecule that migrates to cytoplasm.
5. 4 cAMP bind to 2 regulatory subunits of protein kinase A (PKA).
6. PKA catalytic subunits are released - phosphorylate a small number of target molecules in cell.

Question 15

Name some well known endocrine and paracrine agonists that act at Gs-GPCRs.

Answer 15

1) Adrenaline (endocrine) - beta adrenoRs
2) Noradrenaline (paracrine) - beta adrenoRs
3) Dopamine (paracrine) - D1 dopamine Rs
4) Histamine (paracrine) - H2 histamine Rs

Question 16

Name some well known endocrine and paracrine agonists that act at Gi-GPCRs.

Answer 16

1. Adrenaline (endocrine) - alpha2 adrenoRs
2. Noradrenaline (paracrine) - alpha2 adrenoRs
3. Dopamine (paracrine) - D2 dopamine Rs
4. Endogenous opioids (paracrine) - Mu opioid Rs

Question 17

What is signal amplification and why is it used?

Answer 17

• A few ligand molecules can result in a very large cellular response - cascade effect.
• Body can respond to small changes.
• E.g. Activation of 1 AC produces many cAMP molecules - can activated many PKA.

Question 18

Describe the chain of events leading to and following from Gq activation.

Answer 18

1. Agonist binds to GPCR - conformational change.
2. GPCR mediates GDP for GTP exchange on G alpha q subunit - dissociation of alpha-GTP from beta/gamma.
3. Alpha q subunit activates phospholipase C.
4. PLC hydrolyses PIP2 into 2 second messengers: diaglycerol (DAG) - remains in PM, and IP3.
   5a. IP3 acts as a ligand for IP3R in ER membrane - calcium release (can increase [calcium] by 5-10 fold).
   5b. DAG activates PKC - phosphorylates own substrates.

Question 19

Name agonists that act at Gq-GPCRs.

Answer 19

• Adrenaline - alpha1 adrenoRs
• Noradrenaline - alpha1 adrenoRs
• Acetylcholine - muscarinic ACh Rs 1 & 3
• Histamine - H1 histamine Rs

Question 20

Describe the GPCR-mediated regulation of inotropy.

Answer 20

1. Adrenaline/noradrenaline bind to beta1 adrenoRs in the ventricles - conformational change.
2. GDP to GTP exchange on G alpha s - dissociates and activates adenylate cyclase.
3. AC mediates ATP to cAMP conversion.
4. 4 cAMP bind to regulatory domains of PKA - dissociate from catalytic domains.
5. PKA phosphorylates VOOC in membrane - allows calcium entry when membrane depolarises.
6. Calcium allows myocytes to contract.

Question 21

Describe the GPCR-mediated regulation of vasoconstriction.

Answer 21

1. Sympathetically released NA binds to alpha1 adrenoRs on arterioles.
2. GPCR mediates GDP for GTP exchange on Gq alpha subunit - dissociates and activates phospholipase C.
3. PLC mediates hydrolysis of PIP2 into IP3 and DAG.
   4a. IP3 acts as ligand for IP3R in SR of smooth muscle cell - release of calcium into sarcoplasm - increased contractibility.
   4b. DAG stays in PM and activates PKC - phosphorylates own substrates - sustains vasoconstriction.

Question 22

Which ligand, GPCR and effectors are used to cause bronchoconstriction?

Answer 22

• Parasympathetically released ACh
• Interacts with mAChR on bronchiolar smooth muscle
• Gq-PLC-IP3/Ca2+ and DAG/PKC pathway

Question 23

Describe the use of GPCRs in the modulation of neurotransmitter release at synapses.

Answer 23

1. Endogenous opioids or analgesics (e.g. Morphine) bind to pre-synaptic mu-opioid GPCRs.
2. Mediates GDP for GTP exchange on Gi alpha subunit - dissociates from beta/gamma.
3. Beta/gamma subunits inhibit VOCCs - reduce Ca2+ influx - reduced NT release from pre-synaptic terminal.

Question 24

How does the cholera toxin interfere with G protein function?

Answer 24

• Inhibits GTPase activity of all Gs alpha subunits (through ADP-ribosylation) - unable to hydrolyse GTP to GDP.
• So G proteins active for 18-24hrs before being degraded - very high cyclic AMP concs.
• Leads to diarrhoea.

Question 25

How does the pertussis toxin interfere with G protein function?

Answer 25

- ADP-ribosylates the Gi alpha subunit - unable to exchange GDP for GTP - G alpha not activated.