What is desensitisation?
Ability of a receptor-mediated response to plateau & then diminish despite sustained agonist exposure.
What is the functional role of desensitisation?
- Enables cells to rapidly respond to changes in agonist concentration over a wide concentration range
- Protects cells from potentially toxic effects of superactivation
Give an example of an oncogenic mutation that illustrates the importance of turning signalling off.
e.g EGF Receptor/HER1 mutation in tumours:
- Glioblastomas-mutant HER1 in which much of the EGF binding domain is deleted - Incapable of binding EGF
- Constitutive dimerisation independent of ligand binding
- Unrestricted trans-phosphorylation & downstream signalling
Describe how Receptor Modification is responsible for desensitisation.
- Commonly involves phosphorylation (GPCRs)/dephosphorylation (RTKs) & inactivation of receptor protein following agonist binding
- Occurs in secs/mins
- RTK dephosphorylation carried out by protein Tyr phosphatases (PTPs)
Describe the RTK activation mechanism.
- Ligand binding promotes receptor dimerisation
- Dimerisation-induced conformational change has 2 effects:
- Allows Mg-ATP to bind to each tyrosine kinase domain
- Allows one of the ATP-bound tyrosine kinases to phosphorylate tyrosines on the other partner in the dimeric complex = Trans-phosphorylation
- RTK Active
Describe how Receptor Sequestration is responsible for desensitisation.
- Internalisation of the receptor away from the cell surface into intracellular endosomal compartments
- Occurs over min/hours
Describe how Receptor Down-Regulation is responsible for desensitisation.
- Degradation of receptors (e.g in lysosomes) following their internalisation
- Occurs after several hours
Describe how Induction of a Receptor-Binding Inhibitory Protein is responsible for desensitisation.
- Activation of signalling triggers gene expression & accumulation of inhibitory proteins that bind & inactivate receptor
- Occurs over hours
Name the 4 mechanisms responsible for desensitisation.
- Receptor Modification
- Receptor Sequestration
- Receptor Down-Regulation
- Induction of a Receptor-Binding Inhibitory Protein
Name the 2 types of Receptor Desensitisation.
Homologous - Receptor-specific
Heterologous - Activation of one receptor desensitises another
What are the 3 steps involved in desensitisation of the ß2-adrenoceptor?
- Multiple mechanisms link receptor phosphorylation
- Receptor sequestration
- Receptor down-regulation
Describe the steps involved in the cAMP-mediated desensitisation of the ß2-adrenoceptor by PKA.
- Ser and Thr phosphorylation of residues in 3rd intracellular loop & C-terminal tail involved in coupling to Gs.
- Blocks receptor/Gs interaction
- Blocks cyclic AMP synthesis
Describe the steps involved in the heterologous desensitisation of the ß2-adrenoceptor by other GPCRs via PKA
Same as cAMP-mediated desensitisation except other Gs-coupled GPCRs activate the adenylyl cyclase.
Describe the homologous desensitisation of the ß2-adrenoceptor by G-Protein-Coupled Receptor Kinases (GRKs).
- Agonist-bound receptor phosphorylated by GRK at Ser & Thr residues near C-terminus
- GRK phosphorylation alone does not desensitise
- GRK phosphorylation triggers binding of arrestin
- Arrestin binding blocks receptor/Gs interaction
Describe what happens when arrestin-binding triggers sequestration of phosphorylated ß2-adrenoceptor.
- Arrestin can simultaneously bind phosphorylated ß2-adrenoceptor & clathrin
[Clathrin = A component of clathrin-coated pits on cell surface]
- Phosphorylated ß2-adrenoceptor clusters in clathrin-coated pits
- Clustered receptor-arrestin complex can internalise & accumulate in endosomal vesicles inside the cell
Describe what happens when arrestin-binding triggers degradation of phosphorylated ß2-adrenoceptors.
- Arrestins can simultaneously bind phosphorylated receptor & NEDD4
- NEDD4 catalyses the polyubiquitylation of Lys residues in cytoplasmic domains of receptor
- Receptor targetted to lysosomes where it is degraded
Describe the 2 phases of Desensitisation.
Rapid Phase: Due to receptor phosphorylation. Recovery takes minutes (receptor dephosphorylation & recycling)
Slow Phase: Due to receptor down-regulation. Recovery takes hours (synthesis of new receptor proteins)
Describe Nephrogenic Diabetes Insipidus (NDI).
- NDI = Inability to concentrate urine in the kidney
- Associated with defective Arg-vasopressin (AVP)/V2 vasopressin receptor signalling (V2 receptor = GPCR)
- Linked to variety of inactivating V2 receptor mutations
- e.g in Arg137→His, mutant receptor constitutively internalised - Unresponsive to extracellular agonist
What mechanism is involved in desensitisation of cytokine receptors?
Induction of proteins that bind to & inhibit signalling from activated receptors.
Describe the JAK-STAT signalling pathway that many cytokine receptors activate.
- Cytokine binding to receptor dimers permits transphosphorylation on Tyr & activation of receptor-associated JAKs (Janus Kinase)
- Active JAKs phosphorylate specific Tyr residues on the receptor's cytoplasmic domain
- SH2 domains on STATS (Signal Transducer and Activator of Transcription) bind to Tyr-phosphorylated receptor & become phosphorylated by receptor-bound activated JAKs
- Tyr phosphorylated STATs form dimers that translocate to the nucleus & initiate transcription of specific target genes
What do STATs trigger the induction of?
Suppressors of Cytokine Signalling (SOCS)
How do STATs trigger the induction of SOCS?
- STAT dimers bind to specific regions within SOCS gene promoters & trigger gene transcription
- SOCS protein binds to activated cytokine receptor & inhibits JAK activity
- Receptor-bound SOCS proteins also catalyse the polyubiquitylation of phosphorylated JAKs
- Degradation of JAKs by the proteasome turns off signalling
How do SOCS proteins inhibit JAK activation? Give an example.
- SOCS SH2 domain binds P-Tyr on activated cytokine receptors.
- KIR (kinase inhibitory region) then interacts with & prevents receptor-bound JAKs from phosphorylating STATs - DESENSITISATION
- SOCS box binds enzyme complex responsible for polyubiquitylation of JAKs - DEGRADATION
e.g SOCS-3 binds to P-Tyr759 on gp130 (signalling receptor for IL-6)
How can distinct P-Tyr residues interact with specific SH2 domains?
Binding site for side chain of amino acid 3 positions down from P-Tyr variable between SH2 domains from different proteins - confers specificity.
What is an example of SOCS-mediated homologous desensitisation?
IL-6 mediated desensitisation of gp130 signalling
Give 2 examples of SOCS-mediated heterologous desensitisation and describe how they induce SOCS-3.
- G-CSF mediated desensitisation of gp130 signalling
- G-CSF can activate STATs & induce SOCS-3 via a different cytokine receptor
- LPS-mediated desensitisation of gp130 signalling
- LPS activates the NF-kappaB pathway which induces SOCS-3 via a STAT-indepedent mechanism
Describe Polycythemia Vera, including what it is associated with, diagnosis, phenotype and most common mutation.
- PV associated with hyperactivation & proliferation of haematopoietic stem cells in bone marrow
- Diagnosis - Hypercellular bone marrow
- Phenotype - Elevated production of platelets, white & red blood cells (elevated haematocrit)
- >90% of cases due to acquired Val617Phe mutation in JAK2
How does the mutation that causes PV induce phenotype?
- Val617Phe JAK2 is constitutively active:
- Phosphorylation/activation of receptors & STATs independent from cytokine binding
- Unregulated downstream signalling
- Val617Phe JAK2 phosphorylates SOCS-3
- Blocks SOCS-3's ability to inhibit JAK2
- Escape from inhibitory regulation
What is Cholangiocarcinoma (CCA), what is it associated with and what have advances in molecular pathogenesis highlighted?
- CCA is highly lethal malignant tumour arising from biliary tract epithelium
- Most patients have uncurable disease at presentation & overall survival rate for <5% of patients is over 5 years
- CCA is associated with chronic inflammation of biliary system
- Advances in molecular pathogenesis have highlighted the importance of epigenetic alterations including SOCS-3 promoter hypermethylation
How does SOCS-3 promoter hypermethylation induce phenotype of CCA?