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PHAR3102 > Regulation of GPCR Signalling > Flashcards

Regulation of GPCR Signalling Flashcards

1
Q

Regulation of Signaling through:

General

A 
1. Hydrolysis from GαGTP to GαGDP ( & RGS
protein family)
2. Receptor desensitization
   ● G-protein receptor kinases
   ● Second messenger dependent kinases
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2
Q

The sites of interaction of the receptor

with other cellular proteins

A

See diagram

D - for dimerisation with other GPCRs
Gs, Gi - for interaction with G-protein
P - phosphorylation sites leading to uncoupling and internalisation
PDZ - for recognising short amino acid motifs
RAMPs - for receptor activity-modifying proteins
GRK - for GPCR kinase
Arr - β-arrestin site
SH2, SH3 - SRC homology, sites for receptor tyrosine kinases

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3
Q

Turning the receptor on

A
  • agonist
  • GTP on, GDP off

signalling pathway, e.g.

  • alpha-q (with GTP bound) binds to PLC beta
  • PIP2 converted into DAG and IP3
  • IP3 causes Ca2+ release
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4
Q

Turn off the signal

A
  1. Rapid termination of the actions of agonists
    e.g. Acetylcholine is degraded by acetylcholinesterase
    Noradrenaline is uptaken back into the neurons
  2. Rapid degradation of the second messengers: e.g.
    phosphodiesterases catalyse the hydrolysis of
    cAMP –> AMP; cGMP –> GMP (Second Messengers lecture)
    3.Intrinsic GTPase activity: Gα subunit hydrolyses
    GTP to GDP + Pi.
  3. Receptor desensitisation
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5
Q

Turn off the signal

  1. Rapid termination of the actions of agonists
A

e.g. Acetylcholine is degraded by acetylcholinesterase
Noradrenaline is uptaken back into the neurons

see diagram

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6
Q

Turn off the signal

  1. Rapid degradation of the second messengers
A

e.g. cAMP and cGMP are inactivated by phosphodiesterases

break cyclic nucleotides to 5’monophosphates

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7
Q

Turn off the signal

3.Gα hydrolyses
GTP to GDP + Pi. (GTPase)

GPCR-mediated activation of heterotrimeric G-proteins

A

GPCR activates heterotrimeric G-proteins when agonist binds

GTPase turns off signalling when it converts GTP back to GDP and Pi, leading to the beta-gamma subunit reassociating with the alpha subunit

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8
Q

Turn off the signal

3.Gα hydrolyses
GTP to GDP + Pi. (GTPase)

Regulation of the G-protein cycle

A

G-protein switch is tightly regulated

Most GTP-binding proteins depend on helper proteins to
modulate the switch of G-proteins between inactive form (GDP)
and active form (GTP):
GEFs, Guanine nucleotide-exchange factors: accelerate the release
of bound GDP from Gα (ligand-bound GPCRs are most important
GEFs for heteromeric G-proteins)
GDIs, Guanine nucleotide-dissociation inhibitors: inhibit the
release of bound GDP to maintain GTPase in an off-state (βγ
subunits function like GDIs)
GAPs, GTPase-activating proteins: stimulate GTP hydrolysis to
inactivate G-protein. (e.g. regulators of G-protein signaling, RGS)

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9
Q

Turn off the signal

3.Gα hydrolyses
GTP to GDP + Pi. (GTPase)

The same regulation principles apply to
small G-proteins

A

The small G-proteins can be switched on (G-GTP) and switched off (G-GDP).
● Rho GTPases are inactive when bound to GDP and are kept in this form by
binding to GDI.
● Rho-GDP exchanges for Rho-GTP, which is catalyzed by GEF (“ON”). In their
active state, Rho GTPases interact with many different downstream effector
proteins, including protein kinases, lipid kinases, phospholipases.
● Rho GTPases hydrolysis to GDP is accelerated by GAP (“OFF”).

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10
Q

Turn off the signal

3.Gα hydrolyses
GTP to GDP + Pi. (GTPase)

RGS proteins

A

Regulators of G-protein signaling proteins.

They are a large family of GAPs. RGS activate GTPases of the Gα subunit to turning off G-protein cascades in ALL eukaryotes. Each RGS protein has preferred Gα that they regulate.

•RGS act as GTP-activating proteins (GAPs) for G proteins
–> limiting the signal generated by GPCRs
•>20 mammalian members
•Conserved RGS domain act as a (GTPase)-activating proteins (GAPs) for Gαi, Gαq and G α12/13
–> lowering the energy required for GTP hydrolysis to proceed

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11
Q

Turn off the signal

3.Gα hydrolyses
GTP to GDP + Pi. (GTPase)

RGS proteins as possible therapeutic targets

A
  • Muscarinic agonists and acetylcholinesterase inhibitors are non-selective.
  • RGS2 inhibitor could increase M1 activities in the CNS, thus increasing cognition-enhancing effects in Alzheimer’s disease patients without increasing M2 induced bradycardia.
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12
Q

Turn off the signal

  1. Receptor desensitisation
A

A major mechanism underlying desensitisation is phosphorylation of the GPCR:

Two main types of GPCR desensitisation:
1. G-protein receptor kinases (GRKs)-mediated, also called (agonist specific) homologous desensitisation, or classical model of desensitisation
2. Second messenger dependent protein kinases mediated e.g. protein kinase A (PKA) and protein kinase C (PKC)
•Negative feedback loop
•Heterologous desensitisation

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13
Q

Turn off the signal

  1. Receptor desensitisation

Steps of desensitisation

A
  1. Phosphorylation of the receptor
    “leading to uncoupling of the receptor from G-protein
  2. Internalisation (sequestration) of the receptor
  3. Down-regulation of total number of receptors
    - decreased synthesis
    - increased degradation
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14
Q

Turn off the signal

  1. Receptor desensitisation

Homologous (agonist specific) desensitisation

A

GRKs preferentially phosphorylate agonist-activated GPCRs

βγ subunits activate GRKs

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15
Q

Turn off the signal

  1. Receptor desensitisation

The classical model

A
  • Phosphorylation: GPCR signalling is attenuated by GRKs-mediated phosphorylation. The phosphorylated receptor interacts with an intracellular protein β-arrestin, which leads to the uncoupling of the receptor from G-protein
  • Internalisation (sequestration): Agonist -occupied receptor is then sequestered into intracellular vesicles by clathrin-mediated endocytosis. The agonist is degraded and the receptor dephosphorylated and returned back to the membrane
  • Down-regulation: After repeated or prolonged activation and desensitisation, receptors can be delivered to lysosomes and degraded after internalisation. This results in a decreased response which is only recoverable with the synthesis of new receptor molecules
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16
Q

Turn off the signal

  1. Receptor desensitisation

GPCR signalling: activation and desensitisation
β-adrenergic receptor signalling in the heart

A

Noradrenaline –> beta-1-AR –> Gs –> adenylyl cyclase –> PKA –> inotropy, chronotropy

or
Noradrenaline –> beta-1-AR –> Gs –> beta-AR kinases 1, 2 (GRK2, 3) / beta-arrestins –> desensitisation –> beta-1-AR

17
Q

Turn off the signal

  1. Receptor desensitisation

Regulation of GRK2

A
  • GRK2 modulates multiple signalling pathways
  • involved in lots of human pathologies when GRK is mutated/modified including chronic heart failure, ovarian cancer, rheumatoid arthritis

GRK2 is regulated by:

  • Actinin
  • phospholipids
  • PKA
  • PKC
  • ERK1/ERK2
  • c-Src
  • RKIP
  • Caveolin
  • Microsomal component
  • Ca2+/calmodulin
18
Q

Turn off the signal

  1. Receptor desensitisation

GRK2 and myocardial contractile function

A
  • catecholamine (CA) binds to beta-AR, causing G-alpha-s to bind to adenylyl cyclase, converting ATP to cAMP, activating PKA, increasing contractility
  • under normal conditions there is a small amount of GRK2 present, which binds to the beta-gamma subunit (?) which causes desensitisation with the aid of beta-arrestin. Therefore, G-alpha-s doesn’t bind to adenylyl cyclase, meaning PKA isn’t activate, resulting in reduced contractility
  • following a cardiac injury, stress leads to increased CA being produced. Increased GRK is also produced leading to significantly uncreased desensitisation and contractility
  • chronically increased beta-AR desensitisation leads to the progression of heart failure
19
Q

Turn off the signal

  1. Receptor desensitisation

Differential signalling pathways activated by β1ARs and β2ARs in cardiomyocytes

A
  • Upregulation of GRK2-β2R activity occurs in heart failure (HF) due to increased chronic sympathetic activity, which appears to cause a pro-death effect
  • GRK2 inhibitor peptide (βARKct) may represent a new treatment against HF
20
Q

Turn off the signal

  1. Receptor desensitisation

Second messenger dependent kinases:
PKA & PKC

A
  • Protein kinase A (PKA)
    activated by cAMP
  • Protein kinase C (PKC)
    activated by DAG
  • Negative feedback loop
  • Heterologous desensitisation
21
Q

Turn off the signal

  1. Receptor desensitisation

Negative feedback loop

A

lecture recording

22
Q

Turn off the signal

  1. Receptor desensitisation

Heterologous desensitisation

A

lecture recording

23
Q

Turn off the signal

  1. Receptor desensitisation

Two state model of agonist-selective desensitisation

A
  • independent states model

- sequential states model

24
Q

Turn off the signal

  1. Receptor desensitisation

Homologous vs heterologous desensitisation

A

Homologous
- Agonist specific
- Stimulation of a particular GPCR leads to
desensitisation of that specific GPCR
- Receptor phosphorylation is mediated by specific GRKs - homologous desensitisation
or by PKA and PKC - negative feedback
- G protein uncoupling is further promoted by the binding of β-arrestins to the agonist-activated GPCRs.
- β-arrestins also act as scaffolding proteins, promoting subsequent internalisation, degradation or resensitisation

Heterologous

  • Agonist non-specific
  • GPCR stimulation leads to desensitisation of other types of GPCRs
  • PKA or PKC indiscriminately phosphorylates receptors that have not been exposed to agonist causing heterologous desensitisation
25
Q

Turn off the signal

  1. Receptor desensitisation

Phosphorylation-independent GPCR uncoupling

A

Phosphorylation-independent GPCR uncoupling can occur

26
Q

Turn off the signal

  1. Receptor desensitisation

Central role of kinase cascades in signal transduction

A
  • when kinase cascades occur as a result of GPCR activation, this can cause desensitisation to occur

see diagram

PHAR3102 (20 decks)

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