G-protein coupled receptors

Question 1

Describe the structure of GPCRs

Answer 1

• there are 7 transmembrane domains
• extracellular N-terminus
• intracellular C-terminus
• 3 extracellular loops (ECL)
• 3 intracellular loops (ICL)

Question 2

what are the 5 superfamilies of GPCRs

Answer 2

• Family A “Rhodopsin” 701
  
  • Most common GPCRs
• Family B “Secretin” 15
  
  • A few peptide hormones
• Family C “Glutamate” 15
  
  • Glutamate and GABA NTs
  • Calcium and some taste receptors
• “Frizzled” 24
  
  • Receptors in development (Wnt)
  • Bitter taste receptors
• “Adhesion” 24
  
  • Cell-cell adhesion and motility

Question 3

Where are the binding sites found within the transmembrane domain bundle?

Name 3 ligands which bind to the rhodopsin family of receptors

Answer 3

the binding sites are found within the transmembrane domain bundle

• adrenaline
• acetylcholine
• dopamine

Question 4

where are the binding sites of glutamate family receptors found

name 3 ligands which bind to glutamate family receptors

Answer 4

glutamate family receptors have a large N terminus called the Venus Fly Trap (VFT) domain, this domain closes around the ligand and activates the receptor

Glutamte, GABA and Calcium ions

Question 5

what are allosteric ligands, and give two reasons why they are therapeutically advantageous?

Answer 5

ligands that bind to a separate site from the agonist orthosteric site

1. Non need to “look” like the orthosteric ligand - large chemokine protein
2. Non-surmountable at high concentrations of chemokine (e.g. inflammation)

Question 6

what is the difference between positive and negative allosteric modulators

Answer 6

Positive allosteric modulators (PAMs) enhance the agonist effect

Negative allosteric modulators (NAMs) inhibit the agonist effect

Question 7

the M2 receptor is an example of a GPCR that uses a positive allosteric modulator to enhance the effect of its orthosteric agonist (Iperoxo), this positive allosteric modulator is LY2119620

where does LY2119620 bind and how does this enhance the binding of ACh and other orthosteric agonists?

Answer 7

LY2119260 binds above the acetylcholine binding pocket, this prevents the dissociation of acetylcholine and other orthosteric agonists enhancing their affinity for the receptor

Question 8

state three properties of allosteric modulators that support the use of allosteric modulators with orthosteric ligands

Answer 8

Increased selectivity

1. Endogenous (native) agonist needs to be present for an effect to be observed (use dependence)
2. Orthosteric binding sites are highly conserved between related receptors, meaning the shape and the amino acids involved are often similar. Allosteric binding sites may be unique to the individual receptor subtypes. meaning we can achieve greater selectivity than orthosteric subtypes

Controlled response

3. The maximal effect of an allosteric modulator is limited (“ceiling” effect). Reduced risk of receptor over-stimulation and on-target side effects.

Question 9

state the conformational changes which occur in steps when an agonist binds to a GPCR and activates it

Answer 9

1. when an agonist binds the extracellular ends of the GPCR, the helices to pull together relative to the plasma membrane
2. as a consequence of this, the intracellular domains open up allowing the agonist bound to be transmitted through the receptor into the cytosol
3. this recruits G-proteins to the receptor where they can be activated

Question 10

what are the 3 subunits of G-proteins

Answer 10

Question 11

once an agonist binds to a GPCR it activates the G-protein cycle

State the steps involved in the G-protein cycle which lead to various effector proteins being activated

Answer 11

1. GPCRs are bound by a Gα subunit which binds GDP and forms a complex with Gβγ
2. when an agonist binds the receptor and activates it, this interaction catalyses the exchange of GDP for GTP on the Gα subunit, activating it
3. as a consequence of this activation Gα-GTP and Gβγ separate
4. Gα-GTP and Gβγ can then go on and activate various effector proteins

Question 12

how does the G-protein cycle then recontinue

Answer 12

1. Gα does have some enzymatic activity so after a while, it converts its bound GTP back to GDP
2. Gα-GDP then reassociates with Gβγ and the cycle continues

Question 13

what are the 5 main GPCR adrenoreceptors for adrenaline and noradrenaline

and which G proteins are they each associated with out of

a) Gq
b) Gi
c) Gs
bonus: state whether they upregulate or down-regulate intracellular cAMP

Answer 13

Question 14

Gs-coupled receptors play an important role in the up-regulation of intracellular cAMP levels respectively

briefly state the steps involved in this up-regulation of cAMP

Answer 14

1. when the receptor gets activated, Gsα-GDP gets phosphorylated to Gsα-GTP
2. Gsα-GTP then diffuses along the plasma membrane until it reaches the effector protein adenyl cyclse
3. Gsα-GTP activates adenyl cyclase by binding to it via the Gsα subunit
4. once activated adenyl cyclase uses ATP as a substrate to produce second messenger cAMP

the production of cAMP is enzymatic meaning multiple molecules of cAMP are produced

Question 15

Gi-coupled receptors play an important role in the down-regulation of intracellular cAMP levels respectively

briefly state the steps involved in this down-regulation of cAMP

Answer 15

1. when the receptor gets activated, Giα-GDP gets phosphorylated to Gsα-GTP
2. Gsα-GTP then diffuses along the plasma membrane until it reaches the effector protein adenyl cyclse
3. Giα-GTP inhibtis adenyl cyclase by binding to it via the Giα subunit
4. this prevents the production of second messenger cAMP

t

Question 16

when cAMP has been produced by the Gs pathway a certain protein senses the increased levels of cAMP and cAMP binds to two of its regulatory subunits activating it, what is this protein?

what does binding to this protein result in?

Answer 16

1. Protein kinase A (PKA)
2. binding to the two regulatory subunits of PKA results in it releasing two of its catalytic subunits which can then go on to phosphorylate target proteins

Question 17

what is the main function of Gq protein-coupled receptors?

how does this result occur?

Answer 17

Gq protein-coupled receptors stimulate phospholipase C to increase intracellular Ca2+

1. when Gqα binds to phospholipase C (PLC) it hydrolyses a plasma membrane lipid called (PIP₂)
2. as a consequence of PIP₂ hydrolysis, it releases two compounds IP₃ and DAG
3. DAG is an effector protein which recruits Protein kinase C (PKC), which then phosphorylates target proteins. IP₃ diffuses into the cytoplasm where it binds to intracellular Ca2+ stores in the endoplasmic reticulumn causes the calcium channels to open and release Ca2+

Question 18

true or false

sustained or repeated exposure to an agonist leads to increased responses

Answer 18

FALSE

• Sustained or repeated agonist exposure often leads to reduced responses (all types of receptor

Question 19

β-arrestins are proteins which can contribute to desensitisation

state two ways in which they can do this

Answer 19

Uncoupling from the G protein

1. GPCR is phosphorylated causing the binding of GRK kinase
2. GRK recruits a protein called b-arrestin
3. b-arrestin stops GPCR from coupling to the G protein by binding in an area that hinders access for the G protein (desensitisation seconds to minutes)

Receptor removal from cell surface

• Arrestins also have protein-protein interaction points on the arrestin protein
• arrestins guide the receptor to clathrin coated pits, which are the sites for endocytosis of vesicles away from the plasma membrane
• So the receptors are target to the clathrin-coated pits and undergo endocytosis
• (internalisation - minutes) Into intracellular compartments called endosomes

Question 20

how can internalised receptors alter receptor numbers?

what effect does this have on receptor reserve?

Answer 20

internalised receptors can be degraded by lysosomes, this decreases the total receptor number

as a result receptor reserve goes down, and with it the agonist potency and maximal response