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Flashcards in Receptor-Response Coupling 2 Deck (30):

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:
  1. Allows Mg-ATP to bind to each tyrosine kinase domain
  2. 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.

  1. Receptor Modification
  2. Receptor Sequestration
  3. Receptor Down-Regulation
  4. Induction of a Receptor-Binding Inhibitory Protein


Name the 2 types of Receptor Desensitisation.

  1. Homologous - Receptor-specific
  2. Heterologous - Activation of one receptor desensitises another


What are the 3 steps involved in desensitisation of the ß2-adrenoceptor?

  1. Multiple mechanisms link receptor phosphorylation
  2. Receptor sequestration
  3. 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.

  1. Rapid Phase: Due to receptor phosphorylation. Recovery takes minutes (receptor dephosphorylation & recycling)
  2. 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?

  1. STAT dimers bind to specific regions within SOCS gene promoters & trigger gene transcription
  2. SOCS protein binds to activated cytokine receptor & inhibits JAK activity
  3. Receptor-bound SOCS proteins also catalyse the polyubiquitylation of phosphorylated JAKs
  4. 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.

Example 1:

  • G-CSF mediated desensitisation of gp130 signalling
  • G-CSF can activate STATs & induce SOCS-3 via a different cytokine receptor

Example 2:

  • 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?

  • DNA methylation important in stable suppression of gene expression due to enhanced chromatin condensation (epigenetic silencing)
  • Occurs via the addition of a methyl group to the 5- position of the cytosine ring within the context of a C followed by a G (CpG site) 
  • Almost half of the human gene promoter regions contain CpG rich regions (CpG islands)
  • SOCS-3 promoter contains a CpG island that is hypermethylated in CCA - Prevents SOCS-3 induction resulting in sustained IL-6 signalling