Receptor Internalisation and Alternative Signalling Pathways

Question 1

Pathways of receptor mediated endocytosis

Answer 1

• Activated receptors can be internalised from either clathrin-coated pits or caveolae.
• They are then transported into early endosomes.
• From the early endosomes they can be trafficked back to the cell surface or to late recycling endosomes or to late endosomes.
• The late endosomes become multivesicular bodies (MVB).
• The MVB are then trafficked to a lysosome where the receptor undergoes protein degradation.

Question 2

Endocytic trafficking of catalytic receptors

Answer 2

• Signalling receptors can be internalised by either clathrin-mediated endocytosis or non-clathrin-mediated endocytosis.
• RAB and ARF proteins are small GTP-binding proteins that control trafficking
  in the endosomes.
• Ubiquitylation (Ub) of the receptors leads to trafficking from the multivesicular
  bodies to the lysosomes.

Question 3

Activated Receptor Tyrosine Kinases can internalise via clathrin-coated pits and either recycle rapidly via early endosomes or slowly via multivesicular bodies

Answer 3

Epidermal growth factor (EGF) receptor
- internalise with their ligand

Transforming growth factor (TGF) receptor
- TGF-α receptors dissociate
from their ligand in early
endosomes

Question 4

GPCRs can be internalised via clathrin-coated pits and either recycle rapidly via early endosomes or slowly via multivesicular bodies

Answer 4

The same pathways are also used by GPCRs.

Question 5

Internalised α1B-AR is found in Early Endosomes

Answer 5

The α1B-adrenergic receptor is internalised into early endosomes after activation by the agonist epinephrine (Epi). This internalisation can be inhibited by the use of a receptor antagonist (prazosin) or disruption clathrin pit formation.

Question 6

Not all GPCRs internalise after agonist activation.

Answer 6

The α1B-adrenergic receptor is internalised after activation by the agonist epinephrine (Epi).
The α1A-adrenergic receptor is not internalised after activation by the agonist epinephrine.

Question 7

Desensitisation: The Classical Model

Answer 7

1. Desensitisation
2. Sequestration
3. Recycling/downregulation

GPCRs can be categorised as Class A & Class B based on their patterns of internalisation, recycling or degradation, and association with β-arrestin

Question 8

GPCRs can be categorised as Class A & Class B based on their patterns of association with β-arrestin

Answer 8

Class A: no colocalisation with β-arrestin in endosome

Class B: colocalisation with β-arrestin in endosome

Question 9

C-terminal Tail Involved in Receptor Mediated β-arrestin Trafficking

Answer 9

To test the hypothesis that the sequence of the c-terminal tail of the receptor is mediating the sustained binding of β-arrestin, the tails of the β2-AR and V2R were swapped.

Switching the c-terminal tails of the β2-AR and V2R swapped their β-arrestin interactions e.g. β2-AR-V2R Cterm co-localises with β-arrestin in the endosome which wild-type β2-AR doesn’t.

Question 10

Class A and B recycling

Answer 10

Class A GPCRs rapidly recycle to cell surface

Class B GPCRs slowly recycle or are degraded

Question 11

Regulation of GPCR Trafficking in the Endosomal-Lysosomal System by
Ubiquitination

Answer 11

• β-arrestin binds to AP-2 and clathrin allowing internalisation of the GPCR.
• This then allows the binding of the ubiquitin ligase Mdm2.
• Mdm2-mediated ubiquitination of β-arrestin is necessary for GPCR internalisation.
• GPCRs can also be ubiquitinated, by ubiquitin ligases such as Nedd4.
• Receptor ubiquniation is required for receptor degradation but not internalisation.

Question 12

The ubiquitin system and endocytosis

Answer 12

• Recruitment of E2 ubiquitin ligase leads to ubiquination of the receptor
• This recruits endocytic proteins such as EPS15, epsin, and HRS.
• These adaptors, in turn, are ubiquitinated by NEDD4

Question 13

Regulation of GPCR Trafficking in the Endosomal-Lysosomal System by Ubiquitination

Answer 13

• Activation of Class A GPCRs induces a conformational change that favours binding of deubiquitinases to βarrestin.
• The removal of the
  ubiquitin promoting
  dissociation of β-arrestin
  from the receptor and
  produces a transient ERK
  signal
• Activation of Class B GPCRs, induces a to β-arrestin conformation that does not favour deubiquitinase binding.
• The presence of the
  ubiquitin tags results in
  prolonged downstream signaling and internalisation of the receptor-arrestin complex and targets the complex for degradation

Question 14

The role of the endosome in signalling

Answer 14

Endosomes are key sites of signalling. They have a dual role in signalling:

(i) they modulate the signalling that originates at the plasma membrane
(ii) they generate unique signalling pathways that don’t function at the plasma membrane. This provides for signal diversification and specificity.

Question 15

The signalling endosome

Answer 15

Internalized TGF-βR interacts with the IP3-binding protein SARA and phosphorylates the SARA-associated molecule SMAD2, promoting dissociation of SMAD2 and its interaction with SMAD4. The
SMAD2−SMAD4 complex then translocates to the nucleus, where it alters transcription.

Question 16

The role of β-arrestin in non-G-protein mediated signalling

Answer 16

Multifaceted functions of beta-arrestins

Desensitisation

• G-protein uncoupling
• 2nd messenger degradation

Endocytosis

• Internalisation
• Intracellular trafficking: recycling or lysosomal degradation

Signalling

• Chemotaxis
• Apoptosis
• Metastasis
• Protein translation

β-arrestins act as to prevent G protein activation by the receptor, and as scaffold proteins for a variety of proteins facilitating internalisation and signalling.

Question 17

β-Arrestins function as versatile adaptors for different cell-surface receptors

Answer 17

• For GPCRs, βarrestins act as
  clathrin–AP2 adaptor as well as an E3 ligase adaptor, facilitating internalisation and ubiquitination/deubiquitination
• For the insulin-like growth factor receptor (IGF-1R), β-arrestin 1 functions as a clathrin adaptor as well as an E3 ligase adaptor, facilitating internalisation and ubiquitination
• Transient receptor potential vanilloid 4 (TRPV4) channel complexes with the angiotensin 1a receptor (AT1aR), and upon AT1aR activation becomes ubiquitinated in a βarrestin 1-dependent manner
• Na+/H+ exchanger channel NHE1 is internalised and ubiquitinated in a β-arrestin-dependent manner

Question 18

G-protein–dependent ERK activation

Answer 18

Stimulation of G proteins activates the Raf family proteins through several convergent pathways.

Activated (phosphorylated) ERK translocates to nucleus when activated by this pathway

β-arrestin binding to phosphorylated receptors stops G-protein–dependent signalling, while initiating new waves of signal transduction by activating ERK1/2. Activated ERK (pERK) bound to receptor bound-β-arrestin doesn’t translocate to the nucleus

see diagrams

Class A: cell proliferation of pERK
Class B: no nuclear translocation of pERK

Question 19

Receptor internalisation is required for β-arrestin (non G protein/G protein independent) ERK1/2 activation

Answer 19

Experiment
The β2-AR internalises with agonist stimulation
The α2-AR does not internalise with agonist
stimulation

Both showed increased pERK1/2 when stimulated

• α2-AR activation of ERK1/2 is G-protein dependent
• β2-AR activation of ERK1/2 is predominantly G-protein independent

Question 20

The role of β-arrestin in the signalosome

Answer 20

β-arrestin acts as a scaffold
for a number of kinases
(not just ERK)

Question 21

G-protein signalling vs

Non-G-protein (arrestin) signalling speed

Answer 21

G-protein signalling is rapid and acute

• rapid onset
• wanes with desensitisation
• 2nd messenger dependent

Non-G-protein (arrestin) signalling is slow but sustained

• slower onset
• sustained duration
• signalsome-dependent

Question 22

Class a vs b to distinguish

Answer 22

• where ERK2 is localised
• beta arrestin and where it is/what it’s doing

defining feature is about what the beta arrestin does
- both beta arrestin and the activated JNK3 or ERK2 are in the endosomes
therefore class B