Regulation of GPCR Signalling Flashcards
Regulation of Signaling through:
General
1. Hydrolysis from GαGTP to GαGDP ( & RGS protein family) 2. Receptor desensitization ● G-protein receptor kinases ● Second messenger dependent kinases
The sites of interaction of the receptor
with other cellular proteins
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
Turning the receptor on
- 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
Turn off the signal
- Rapid termination of the actions of agonists
e.g. Acetylcholine is degraded by acetylcholinesterase
Noradrenaline is uptaken back into the neurons - 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. - Receptor desensitisation
Turn off the signal
- Rapid termination of the actions of agonists
e.g. Acetylcholine is degraded by acetylcholinesterase
Noradrenaline is uptaken back into the neurons
see diagram
Turn off the signal
- Rapid degradation of the second messengers
e.g. cAMP and cGMP are inactivated by phosphodiesterases
break cyclic nucleotides to 5’monophosphates
Turn off the signal
3.Gα hydrolyses
GTP to GDP + Pi. (GTPase)
GPCR-mediated activation of heterotrimeric G-proteins
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
Turn off the signal
3.Gα hydrolyses
GTP to GDP + Pi. (GTPase)
Regulation of the G-protein cycle
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)
Turn off the signal
3.Gα hydrolyses
GTP to GDP + Pi. (GTPase)
The same regulation principles apply to
small G-proteins
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”).
Turn off the signal
3.Gα hydrolyses
GTP to GDP + Pi. (GTPase)
RGS proteins
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
Turn off the signal
3.Gα hydrolyses
GTP to GDP + Pi. (GTPase)
RGS proteins as possible therapeutic targets
- 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.
Turn off the signal
- Receptor desensitisation
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
Turn off the signal
- Receptor desensitisation
Steps of desensitisation
- Phosphorylation of the receptor
“leading to uncoupling of the receptor from G-protein - Internalisation (sequestration) of the receptor
- Down-regulation of total number of receptors
- decreased synthesis
- increased degradation
Turn off the signal
- Receptor desensitisation
Homologous (agonist specific) desensitisation
GRKs preferentially phosphorylate agonist-activated GPCRs
βγ subunits activate GRKs
Turn off the signal
- Receptor desensitisation
The classical model
- 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
Turn off the signal
- Receptor desensitisation
GPCR signalling: activation and desensitisation
β-adrenergic receptor signalling in the heart
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
Turn off the signal
- Receptor desensitisation
Regulation of GRK2
- 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
Turn off the signal
- Receptor desensitisation
GRK2 and myocardial contractile function
- 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
Turn off the signal
- Receptor desensitisation
Differential signalling pathways activated by β1ARs and β2ARs in cardiomyocytes
- 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
Turn off the signal
- Receptor desensitisation
Second messenger dependent kinases:
PKA & PKC
- Protein kinase A (PKA)
activated by cAMP - Protein kinase C (PKC)
activated by DAG - Negative feedback loop
- Heterologous desensitisation
Turn off the signal
- Receptor desensitisation
Negative feedback loop
lecture recording
Turn off the signal
- Receptor desensitisation
Heterologous desensitisation
lecture recording
Turn off the signal
- Receptor desensitisation
Two state model of agonist-selective desensitisation
- independent states model
- sequential states model
Turn off the signal
- Receptor desensitisation
Homologous vs heterologous desensitisation
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
