GPCRs

Question 1

give the 3 basic domains of a GPCR

Answer 1

1. 7 transmembrane domains
2. ligand binding domain (extracellular)
3. G protein binding domain (intracellular)

Question 2

what is the structure of:

1. family 1A
2. family 1B
3. family 1C
4. family 2
5. family 3

Answer 2

1. contains a short N terminus; ligand binding domain is buried within the TM domains
2. ligand binding domain is on the surface, allowing it to recognise bigger ligands than family 1A
3. has a long N terminus; recognises very large ligands such as hormones
4. ligand interracts with N terminus; is far removed from other families
5. v long N terminus with clam like structure that traps ligands. Functions as a dimer.

Question 3

describe the 4 stages of signal transduction

Answer 3

1. ligand binding induces a conformational change in the receptor
2. GPCR acts as a GEF, exchanging GDP to GTP in the associated G protein’s alpha subunit
3. the alpha and beta subunits dissociate and transduce the signal to downstream effectors
4. GTP is hydrolysed, therefore the alpha and beta-gama subunits reassociate and signalling is terminated.

Question 4

1. Describe the basis of the FRET assay
2. what did a FRET assay show about the interraction between G proteins and GPCRs?
3. What are the implications of these interractions?

Answer 4

1. BFP is added to one protein. YFP is added to another. Light of 436nm excited BFP, which emits lights at 480nm. YFP is excited at 480nm, and emits light at 536nm.

If the proteins that are tagged with BFP and YFP are within 10nm of each other, (i.e. associated), then the light emited by excited BFP will excite YFP, thus overall, 2 wavelengths of light (480nm and 536nm) will be emited, revealing 2 colours. If they are not associated, then only blue will be seen.

2. in the absense of a ligand, only blue was seen, suggesting that the GPCR is not pre-coupled to a G protien, and coupling only occurs upon ligand binding.
3. multiple G proteins have the potential to couple to the activated GPCR, therefore numerous signaling outcomes may be initiated.

Question 5

1. what does the cannonical signalling pathway involve?
2. what does the non-canonical pathway involve?
3. to discover whether coupling of B2AR could switch between G proteins, how was activation of the non-canonical pathway measured?
4. cells were preincubated with the pertussis toxin. what is the effect of this, and why was it done?
5. what does βARKct do? Why was this used?
6. What is H-89?
7. What happened to pMAPK levels upon ligand binding?
8. what was the result of using a phosphodeficient β2AR?
9. what was the effect of the pertussis toxin on pMAPK levels?
10. what was the effect of βARKct on the pMAPK levels?
11. what was the effect of H-89 on pMAPK levels?
12. What was the overall conclusions of these experiments?

Answer 5

1. activation of adenylyl cyclase, to increase cAMP levels leading to PKA activation
2. activation of MAPK module.
3. western blotting, to measure pMAPK levels in response to agonist binding
4. it inhibits Gi signalling, by adding ADP ribose to Gi thus inactivates Gi signalling
5. it sequesters all the βγ subunits, to see whether they are involved in transducing the signal to MAPK
6. a PKA inhibitor
7. increased
8. the increase in pMAPK levels in response to ligand binding was abolished (suggesting that the receptor must be phosphorylated for MAPK to become activated)
9. prevented increase in pMAPK (Gi transduces signal to MAPK)
10. prevented increase in pMAPK (βγ subunits transduce signal to MAPK)
11. prevented increase in pMAPK
12. ligand binding > Gs > adenylyl cyclase > increase cAMP > PKA activation > PKA phosphorylates receptor > Gi > βγ > sos > ras > MAPK

changes in GPCR phosphorylation status changes which G protein associates, therefore influences downstream signalling events.

Question 6

Name 6 factors which regulate specific GPCR signalling in a context specific manner.

Answer 6

1. lipid microdomains
2. scaffold proteins
3. adaptor proteins, which enable G proteins to signal indirectly to proteins
4. splicing
5. post-translational modifications
6. endocytosis - internalised GPCRs can continue to signal, but the signalling pathway might be different to that on the cell surface.

Question 7

Describe the process of homologous desensitisation involving GRKs and Beta arrestin.

Answer 7

1. following prolonged receptor activation, GRKs are recruited to and phosphorylate the receptor
2. p-GPCR binds β-arrestin; this prevents the reassociation of G proteins
3. β-arrestin couples p-GPCR to the endocytic machinery
4. in the endosomes, the low pH causes the ligand to dissocate from the receptor. This causes arrestin to fall off.
5. the resensitised receptor is recycled back to the plasma membrane.

Question 8

1. which 2 GRKs and which 2 arrestins function in the retina?
2. what are the effects of knocking out GRK3 or Arrestin 3?
3. what are the C and N terminals of beta arrestin separated by?
4. what does the C terminal of β-arrestin bind to?
5. what are class A receptors? Give example
6. What are class B receptors? give example

Answer 8

1. GRK1 and 7; arrestin 1 and 4
2. morphine is a more potent analgesic with fewer side effects.
3. phosphodetection domain (recognises phosphorylated GPCRs)
4. heavy chain of clathrin and AP2 (endocytic machinery)
5. receptors which associate transiently with βarrestin, and are rapidly recycled. e.g. β2AR
6. receptors which associate for a long time with βarrestin and are slowly recycled. e.g. V2 vasopressin receptor.

Question 9

1. what is the phosphorylation barcode?
2. how can different GRKs be engaged?
3. what is βarrestin recruitment regulated by?
4. what is the strength and duration of βarrestins interraction with the receptor governed by?
5. name 1 problem with the phosphorylation barcode theory.

Answer 9

1. different sets of GRKs phosphorylate distinct sites on GPCRs; which sites are phosphorylated is dependent upon which GRKs are recruited, and constitutes a barcode.
2. by the conformation of the receptor that is ligand dependent. For example, the conformation induced by one ligand will engage different GRKs to the conformation induced by another ligand. The conformations induced by a ligand will also determine which G proteins can be recruited.
3. the subset of receptor phosphorylation sites
4. patterns of receptor phosphorylation and ubiqutination
5. it is yet to be experimentally proven.

Question 10

1. what is tolerance?
2. How has tolerance to morphine been shown in vivo?
3. Name the general method that were used to test whether trafficking and desensitisation had any role in tolerance?
4. what happened when DAMGO was applied (in terms of arrestin translocation)
5. what happened when morphine was applied (in terms of arrestin translocation)
6. what were used to observe receptor internalisation upon the application of a ligand. What were the effects of DAMGO and morphine application on this?
7. what were the effects of expressing a dominant negative GRK that is unable to phosphorylate receptors?
8. what were the effects of inhibiting PKC?
9. what is the overall conclusion of these experiments?

Answer 10

1. the reduction on efficacy of a drug with repeated exposure.
2. naive subjects show a more effective response to pre-exposed subjects when given the same dose
3. fluorescently labelling arrestin to observe translocation to the plasma membrane.
4. translocation of arrestin to plasma membrane
5. no translocation of arrestin to the plasma membrane.
6. ELISA and confocal microscopy. DAMGO induced receptor internalisation, while morphine did not.
7. prevents desensitisation to DAMGO, but not desensitisation to morphine
8. abolished morphine desensitisation but not DAMGO desensitisation.
9. morphine desensitsed receptors are phosphorylated by PKC, therefore are not internalised thus resensitised. DAMGO desensitised receptors are phosphorylated by GRK, therefore are internalised thus resensitised.

Question 11

1. What does βarrestin have binding sites for? (4)
2. what is the first phase of ERK activation dependent upon?
3. what is the second phase of ERK activation independent of?
4. Which phases are sensitive and insensitive to H-89?
5. What phase was blocked in the Gs KO?
6. What is the TYY mutant?
7. What do these findings suggest?
8. Which other ligands show signalling bias?
9. How does G-protein independent signalling impact on traditional drug screens?

Answer 11

1. GAPs, GEFs, GTPases and MAPKs
2. Gs and PKA
3. G proteins
4. 1st phase is H-89 sensitive; second phase is insensitive to H-89
5. 1st phase
6. a β2AR mutant that abolishes g protein activation, but leaves beta arrestin signalling intact.
7. ERK activation can be activated independently of G proteins via βarrestins.
8. DAMGO and morphine. Morphine shows a beta arrestin bias, while DAMGO shows a G protein bias
9. traditional screens involve analysis of second messengers, meaning only the effects on G protein signalling will be detected. New screens looking for the effects on Beta arrestin signalling are therefore necessary.

Question 12

Using the μOR and angiotensin II receptors as examples, suggest how biassed ligands may be better drugs.

Answer 12

1. at the μOR, G protein signalling s assocated with analgesia, while beta arrestin signalling is associated with side effects such as constipation and respiratory depression. Using a G protien biassed ligand as a drug could provide analgesia without the side effects
2. at angiotensin II receptors, beta arrestin signalling produces increased cardiac contractility and decreased cardiac cell death, while Gq signalling produces side effects such as fluid retention. Therefore it is beneficial to produce a beta arrestin biassed ligand.

Question 13

highlight 3 problems faced whrn trying to crystalise the GPCR structure.

Answer 13

1. integral membrane proteins don’t fold properly outside of membranes
2. they are expressed in low levels which makes purification difficult. Large amounts of pure protein is required for purification
3. GPCRs are highly dynamic proteins. Dynamic structures don’t crystalie properly.

Question 14

1. What are the 2 most dynamic structures of the GPCR?
2. How were these structures engineered to make the receptor more stable and suitable for crystalisation?
3. was the functionality of this engineered receptor any different from the native receptor?
4. what were inverse agonists used for?
5. could the engineered receptor still adopt an active conformation?
6. Name 2 other methods for stabilising the receptor.

Answer 14

1. the IC loop and the C-terminus
2. C terminus was removed. IC loop was replaced with T4 lysosyme, which is of the same size, still links the 2 TM domains and is much more stable.
3. No
4. to stabilise the receptor in an inactive conformation
5. yes
6. use of antibodies to improve crystal lattice forming contacts; by introducting multiple mutations throughout the receptor

Question 15

1. what is the conformation of the EC part of the receptor?
2. What was found about the transmembrane domains of the receptor?
3. interractions with what contribute to receptor clustering into microdomains?
4. displacement of what enables access to the ligand binding pocket?

Answer 15

1. open, to allow for the enrty and binding of ligands
2. kinks
3. cholesterol
4. EC loop 2

Question 16

1. Name 4 GPCR structures which were compared to one another.
2. in which structures were the biggest divergences seen?
3. in which 2 receptors were the ligand binding pockets highly conserved? What does this explain?
4. What can be used to explain differences between basal activity of 2 members of the same subfamily?

Answer 16

1. β1AR, β2AR, A2A and Rhodopsin
2. EC loops and ligand binding pocket
3. β1AR and β2AR. Explains the difficultly in developing a potent selective drug
4. the IC loop of β1AR is more rigid than that of β2AR

Question 17

1. what is the conformation of the opiod receptor’s ligand binding site? Why is this conformation necessary?
2. What evidence suggests that opoid receptors function as dimerisation?

Answer 17

1. relatively open conformation. Must be to accomodate a wide variety of ligands
2. they tend to crystalise together.

Question 18

1. what does Gs activate?
2. what does Gi activate/inhibit
3. what does Gq activate?
4. what is GTP hydrolysis regulated by?
5. how does PKA/PKC receptor phosphorylation promote desensitisation?
6. How does GRK receptor phosphorylation promote desensitisation?

Answer 18

1. adenylyl cyclase
2. inhibits adenylyl cyclase, activates K_ir
3. PLCβ
4. RGS
5. acts as a classical negative feedback loop by uncoupling the receptor from the G protein
6. leads to homologous desensitisation and β-arrestin recruitment.

Question 19

1. what proteins organise MAPK pathways? How do they do so?
2. How does β-arrestin act as one of these proteins and for which pathways?
3. What does β-arrestin activation of ERK contribute to?
4. β-arrestin is important in the transduction of which signals?

Answer 19

1. SCAFFOLD PROTEINS - they tie together appropriate kinases in a series forming a discrete signalling module
2. it binds all of the kinases of the ERK1/2 activating module (Raf, Mek, Erk) and the JNK activating module.
3. anti-apoptotic signalling
4. chemoattractants.

Question 20

1. give a piece of evidence for receptor dimerisation
2. is dimerisation essential in GPCR signalling?
3. state 3 roles that receptor dimerisation plays

Answer 20

1. β2AR could be co-immunoprecipitated from cell membranes
2. no
3. post translational processing; trafficking to the PM; generating functional diversity.

Question 21

1. full agonist
2. partial agonist
3. inverse agonist
4. antagonist

Answer 21

1. maximally activate receptor
2. produce submaximal activity, even at saturating concentration
3. inhibit/decrease basal activity of the receptor by holding it in an inactive conformation
4. has zero efficacy for the receptor.

Question 22

1. how many amino acids differ in the binding pockets of β1AR and β2AR
2. what are the similarities betweren the residues that line the interiors of β1AR and β2AR
3. how many differences are there in the binding pockets of β2AR and α2AR

Answer 22

1. 1 out of 15
2. 4
3. they are identical

Question 23

how has the crystal structure of β2AR impacted GPCR drug discovery?

Answer 23

in silico screens have enabled the identification of new ligands

Question 24

1. How is opiod induced analgesia mediated?
2. why is morphine a more potent analgesic in β-Arrestin KOs?
3. Name a g protein biassed ligand for the opoid receptor.

Answer 24

1. g proteins inhibit ion channels, leading to hyperpolarisiation of nociceptive fibres.
2. beta arrestin inhibits the g protein mediated response.
3. TRV130