In the notch pathway, ___ and Jagged are ___.
Notch is composed of ___.
- NECD - Notch extracellular domain
- transmembrane domain
- NID - Notch intracellular domain
___ cleaves the NECD from the ___.
What is ADAM?
A desintegrin and metalloprotease
The NICD makes its way to the ___ and generally results in ___.
- transcription, cell division, differentiation
Hh are ___, a ___.
- family of secreted proteins
Hh generally functions in ___.
In adult cells Hh can lead to ___.
Hh homologues in vertebrates include ___.
sonic, desert, Indian Hh
What is the receptor for Hh?
What are the receptors for Hh in mammals?
Besides Patch and Hip, what else is needed for Hh pathway activation?
The ultimate target of Hh in the fruitfly is ___, a ___.
- cubitus interruptus (Ci)
- transcription factor
What vertebrate homologue most closely resembles Drosophila Hh?
How do Shh, Dhh, and Ihh differ?
Typically by potency - Shh>Ihh>Dhh
What role in develoment does Hh play?
cellular proliferation, growth, and axon path finding
What are examples of human developmental disorders from Hh?
- Greig cephalopolysyndactyly syndrome
- Pallister-Hall Syndrome
- Gorlin's syndrome
What are some cancers triggered by malfunctioning Hh?
- Basal cell carcinoma
- Small cell lung cncer
- Pancreatic cancer
What are some components of the Hh pathway?
- Patch(Ptc) - membrane receptor
- Smoothened (Smo) - intermembrane protein
- Intracellular Hh Signaling complex (HSC)
- Membrane receptor which activates Smo when bound to Hh.
- After binding, Ptc levels decrease as a result of endocytosis
In vertebrates, Ptc does not have ___, so it needs ___.
Intermembrane protein that when activated relays signals to HSC
In vertebrates, Smo is always ___.
at the cell membrane
- Coastal 2 (Co2) - kinesin-related protein
- Fused (Fu) - Ser/Threo kinase
- Supressor of fused (Su/Fu)
- Cubitus Interruptus (Ci)
What are three Ci homologs as activators in mammals?
Gli1, Gli2, Gli3
Since Gli acts only as an activator, it does not get ___.
What happens to Ci when there is no Hh?
HSC truncates Ci which becomes a repressor
What happens when Hh binds to Ptc?
Production of Ci which becomes an activator
In the Drosophila wing imaginal disc, Ci is truncated where?
In all but cell fate 1 nearest the posterior
In the Drosophila wing imaginal disc, Hh concentration results in activation where?
In all but cell fate 5 (nearest the Anterior) which results in repressor.
In the Drosophila wing imaginal disc, how does Hh concentration affect expression?
[Hh] is proportional to activation
What is involved in Hh processing?
- Binding of cholesterol to C end
- Addition of palmitate to amino terminus
Describe the binding of Hh to cholesterol.
- Critical for target cell intake
- Critical for signal transduction after Hh binds to Ptc
- If binding is inhibited, Hh doesn't work
In the absence of Hh, Ptc ___.
blocks the phosphorylation and stability of Smo.
What type of receptor does Smoothened have?
G protein coupled receptor
When there is no Ptc, ___.
Smo is found in endosomal vesicles
Upon Hh binding to Ptc, ___.
Smo is released and localizes to the cell membrane
Smo multimers may be required for ___.
high level signaling
With Hh present, Ci__ is processed to ___ or ___.
- Weak activator - Ci^act
- Strong activator - Ci*
If no Hh is present, Ci__ is formed.
How is Ci phosphorylated?
- Glycogen synthase Kinase 3-beta
- Casein Kinase 1-gamma
Upon phosphorylation, Ci is ___ by ___, a ___.
- Slimb (supernumerary limbs)
- proteosome for cleavage
Describe Coastal 2 (Cos2)
- Possibly a microtubule-motor
- Interacts with Smo
- Responsible for moving Smo
How do Cos2 and Smo interact?
- Cos2 binds Fu to Smo
- Their interaction is critical for hh pathway signalling
Where does Cos2 move Smo?
- cell membrane upon Hh pathway activation.
- Intracellular vessicles in the absence of Hh ligand
Describe Fused (Fu)
- Has kinase activity, i.e. might phosphorylate Cos2 and Su(Fu) upon Hh pathway activation.
- Binds to Cos2 and Su(Fu) via carboxy terminus domain
Describe Suppressor of Fused
- May be antagonistic to Fu
- No a.a. homology to known proteins
- Binds Fu and Ci, but not Cos2
What evidence is there that Su(Fu) and Fu might be antagonistic?
Su(Fu)- and Fu- flies yield a wt phenotype
What are possible functions of Su(Fu)?
- May inhibit Ci activation
- Nuclear translocation of Ci
- Transcriptional regulation in vertebrates
Describe Hh pathway at no/low [Hh].
- Ptc on cell membrane repressing/sequestering Smo at vesicle with HSC-A inactive.
- HSC-R on MT picking up vesicle leading to truncated Ci^75 by Su(Fu).
- Ci^75 to nucleus as repressor
Describe Hh pathway at medium [Hh].
- Hh binds Ptc at cell membrane permitting Smo/HSC-A to go to cell membrane
- HSC-R not at MT
- HSC-A allows Ci^act into Nucleus for low activation with possible low amount of Ci^75.
Describe Hh pathway at high [Hh].
- Hh sequesters Ptc to vesicle permitting Smo Multimer/HSC-A to go to cell membrane
- Fu phosphorylates Cos2/SuFu
- SuFu leaves HSC
- Cos2 drives Ci to Smo
- HSC-R not present/inactivated.
- HSC-A allows Ci* to be processed untruncated into Nucleus by SuFu for high activation
Research shows that Hh signalling malfuction is responsible for:
- Formation of tumors
- Survival of tumors
In mammals, absence of Hh leads to ___.
- Gli forms MT-attached complex with Fu and SuFu
- Gli remains in cytoplasm
In mammals, if Hh is present, ___.
- Hh binds to Ptc
- Smo is activated (no longer supressed)
- Processing of Gli is activated
- Gli is translocated to nucleus
In mammals, the negative feedback regulators in the Hh pathway are ___.
Ptch, Hip, Gli
What genes are for cell proliferation?
- Cyclins D1 and D2 (mammalian) -> mitosis
What are three target proteins?
- Cyclin B - Mitotic P Factor (MPF) - nuclear translocation
- P21 inhibition - tumor supression
- PDGF pathway activation (MAPK) - cell division
What are two types of genetic problems w.r.t. cancer?
- LOF for tumor supression - Ptc and SuFu
- Overexpression of oncogenes - Shh and Smo
LOF of SuFu leads to ___.
LOF of Ptc1 leads to ___.
cell nevus carcinoma
Heterozygous Ptc mice ___.
Blocking Smo blocks ___.
binding of Hh
What should be inhibited for cancer treatment?
What are inhibitors of Smo?
- Cyclopamine - binds to Smo, but difficult to synthesize and toxic
- KAAD - modified cyclopamine - less toxic
How do you inhibit Gli?
- Forskolin - PKA activator - used in different pathways as well
- RNAi - Not feasible for treatment