Cell signalling 4: signalling through receptors II (Prof. Patel)

Question 1

What is the structure of receptors guanylyl cyclases ?

How are they are they activated ?

Answer 1

• enzyme-linked receptors (dimers) composed of an extracellular domain, a transmembrane domain, a kinase homology domain, a linker and a cylase domain
• activated by natriuretic peptides = ANPs (Atrial Natriuretic Peptides) and BNPs (Brain Natriuretic Peptides)

Question 2

What molecule does the activation of receptor guanylyl cyclases produce ?
Is this the only way of producing such a molecule ?

Answer 2

cGMP –> can also be produced by soluble guanylyl cyclases

Question 3

What is the structure of soluble guanylyl cyclases ?
By which ligand are they activated ?
What are the characteristics of this ligand ?

Answer 3

Structure = dimer, made of a heme binding domain, a linker and a cyclase domain
Soluble GCs are activated by nitric oxide (NO) = a gas –> cell-permeable, short-lived ==> gives rise to a “local” signal

Question 4

How are cGMP levels reduced in the cell ?

Answer 4

By phosphodiesterases (w/ H2O) which convert cGMP to GMP.

Question 5

Which enzyme mediates many of the effect of cGMP ?

Answer 5

PKG (made of a R and C domain like PKA).

Question 6

What are the physiological effects of NO/cGMP signalling ?

Answer 6

NO/cGMP signalling regulates smooth muscle relaxation
1. When stimulated by ACh, endothelial cells (cells lining the interior of blood/lymphatic vessels), activate NO synthase which converts Arg to NO which can diffuse across the membrane towards smooth muscle cells
Neurons can also directly release NO
2. NO can bind to soluble GCs, which lead to conversion of GTP to cGMP
3. cGMP activates PKG, which:
- causes Ca2+ to be pumped back into the ER
- stimulates K+ efflux
- inhibits Ca2+ influx
- inhibits PDE-S (Phosphodiesterase-S)
4. Overall decrease in [Ca2+] leads to smooth muscle relaxation

Question 7

Who won the Nobel Prize in Medicine “for their discoveries concerning NO as a signalling molecules in the cardiovascular system” in 1998 ?

Answer 7

Robert F Furchgott, Louis J Ignarro and Ferid Murad.

Question 8

By which ligands are receptor serine/threonine kinases activated ?
What are the two type of receptor serine/threonine kinases ?

Answer 8

• TGF-beta (Transforming Growth Factor-beta)
• Activin
• BMP (Bone Morphogenetic Protein)
  Type I = phosphorylate specific smad proteins
  Type II = phosphorylate type I receptors

Question 9

What are the best studied receptor serine/threonine kinases ?
What do these signal through ?

Answer 9

The TGF-beta superfamily.

Activated TGF-beta receptors signal through smad proteins.

Question 10

What do phosphorylated smad proteins do ?

Answer 10

Phosphorylated smad proteins asociated w/ smad 4 to regulate gene expression:

• TGF-beta/activin/Nodal –> activate TGF-beta/activin target genes
• BMPs/GDFs (Growth Differentiation Factors) –> activate BMP genes

Question 11

For which embryonic process is TGF-beta (Nodal) signalling important ?

Answer 11

Mesoderm induction.

Question 12

What is Wnt signalling ?

Answer 12

Wingless (Drosophila) + Int-1 (mouse) = Wnt
Wnt signalling = signalling through proteolysis
Wnt = secreted proteins
Signal through:
- Canonical pathway
- Planar cell polarity (PCP) pathway
- Ca2+ pathway

Question 13

What are the different steps of Wnt canonical signalling ?

Answer 13

Wnt = stimulus
Frizzled and Lrp5/6 = receptor
Dishevelled = effector
β-catenin = transcription factor

Question 14

What is the fate of β-catenin when Wnt is not bound to frizzled and Lrp5/6 ?
What about LEF and TCF target genes ?

Answer 14

β-catenin undergoes protelysis by forming a complex w/:
1. Axin
2. APC (Adenomatous Polyposis Coli)
3. GSK3 (Glycogen Synthase Kinase 3)
4. CK1α (Casein Kinase 1α)
LEF and TCF taget genes are normally repressed by Groucho

Question 15

How does Wnt prevent β-catenin proteolysis ?

Answer 15

When Wnt bind to frizzled and Lrp5/6, it recruits dishevelled which “steals” axin and forms a complex w/ frizzled and Lrp5/6 –> less β-catenin proteolysis –> β-catenin levels go up and can displace Goucho –> activate transcritption of LEF and TCF genes

Question 16

Why is Wnt signalling important ?

Answer 16

• embryonic development

- maintenance of nerve circuits

Question 17

What is hedgehog ?

What are the 3 types ?

Answer 17

Hedgehog = secreted signalling proteins

3 types: sonic, indian and desert

Question 18

What are the different steps of Hedgehog signalling ?

Answer 18

Hedgehog = stimulus
Patched = receptor
Smoothened = effector
Cubitus interruptus(Ci) / Gli (mammalian) = transcription factor

Question 19

What is the fate of Ci when Hedgehog is not bound to patched ?
What about when Hedgehog is bound ?

Answer 19

• when Hedgehog is not bound, Ci undergoes proteolysis to repress transcription
  It forms a cocomplex w/:
  1. Co (Costal) (MT binding)
  2. Fu (Fused) = kinase
  3. Sufu (Supressor of fused) = adaptor protein
• when Hedgehog is bound, Ci proteolysis is inhibited –> transcription stimulated

Question 20

What is the effect of smoothened on Ci proteolysis ?
What is the effect of patched on smoothened ?
What can we say about the effect of Hedgehog then ?

Answer 20

Smoothened inhibits Ci proteolysis.
Patch inhibits smoothened.
Thus, when Hedgehog binds patched, it “de-inhibits” smoothened.

Question 21

Why is Hedgehog important ?

Answer 21

Hedgehog signalling regulates the polarity of segments in the embryo.