Signaling Pathways and Development

Question 1

similarities of hedgehog (Hh) and Wnt pathways (3)

Answer 1

regulated by proteolysis

involved in embryonic development

involved in maintaining stem cell niches (implicated in cancer)

Question 2

contrast developmental roles of
Sonic Hh
Indian Hh
Desert Hh

Answer 2

Sonic Hh: CNS
Indian Hh: cartilage and bone
Desert Hh: peripheral nerves

Question 3

what are the transcriptional effectors of Hh family genes

Answer 3

Ci (Drosophila) //
Gli1/2/3 (mouse and human)

Question 4

describe these components of Hh signaling pathway:
a. Hedgehog
b. Patched-1 (Ptc1)
c. Smoothened (Smo)
d. Gli/Ci proteins
e. PKA

Answer 4

a. Hedgehog (Shh, Ihh, Dhh): ligands, secreted hydrophobic proteins produced by localized group of cells (embryos and adults)
b. Patched-1 (Ptc1): 12-TM receptor, binds Hh ligands
c. Smoothened (Smo): 7-TM GPCR (*does NOT bind Hh, is BLOCKED by Ptc1)
d. Gli/Ci proteins: TF that activator or repress target gene expression depending on signaling status
e. PKA: phosphorylates Gli/Ci to target them for proteolysis in proteosome

Question 5

what happens in Hh pathway when Ptc1 is present as receptor

Answer 5

1. Hh ligand binds to Ptc1, preventing it from inhibiting Smo —> Smo is DISinhibited
2. Smo disinhibition prevents PKA phosphorylation of Gli proteins
3. Smo BLOCKS conversion on Gli proteins into repressors —> Gli proteins are DISinhibited to become activators (GliA) of target genes

Question 6

what happens in Hh pathway when Ptc1 is NOT present as receptor

Answer 6

1. Hh cannot bind Ptc1 —> Ptc1 continues to inhibit Smo
2. Gli proteins are phosphorylated by PKA and directed to proteosome
3. C-terminus is cleaved —> Gli proteins are converted into transcriptional repressors (GliR) of target genes

Question 7

how do Shh signaling transduction components localize to the primary cilium (non-motile cilium, sensors of extracellular information)

Answer 7

1. Ptc1 inhibits Smo
2. Gli proteins shuttled in/out of cilium by IFT (intraflagellar transport proteins)
3. Shh ligand binds Ptc1, Smo is disinhibited
4. Smo enters cilium and blocks Gli phosphorylation —> GliA

[IFT move cellular cargo in/out of cilia to cell body along microtubules, using kinesin/dynein]

Question 8

congenital (inherited) human disorders of Hh signaling primarily affect ___ and ____

Answer 8

limb and CNS

Shh is localized to organizing centers in posterior limb bud and ventral midline of CNS during embryogenesis

Question 9

major biological causes (2) of human holoprosencephaly (HPE)

Answer 9

HPE: failure of embryonic prosencephalic vesicle to divide into two hemispheres

major causes:
1. SHH protein haplo-insufficiency (single copy of gene is not sufficient)
2. GLI2 mutation

[Shh knockout (Shh-/-) in mice induces HPE and limb defects, but limb defects NOT seen in humans]

Question 10

what is the mechanism and effect of cyclopamine (plant alkaloid)

Answer 10

inhibits Shh signaling by blocking Smo (which Shh disinhibits by binding Ptc1)

Gli phosphorylation into GliR occurs constitutively (target genes constantly repressed)

Question 11

what is the role of cholesterol in Hh signaling

Answer 11

required for production and secretion of mature Hh protein - needed for self-cleavage reaction that truncates Hh protein precursor

processed form of Hh contains covalent cholesterol adducts in active signaling sequence

Question 12

Smith-Lemli-Opitz Syndrome

Answer 12

mutation in cholesterol synthesis [sterol delta-7-reductase]

cholesterol depletion during gestation —> depleted processing of Shh (needed for auto-cleavage)

phenotypes similar to knock down of Shh (limbs and CNS effects)

[SHHmith-lemLESTEROL syndrome]

Question 13

mutations in this component of Hh signaling are linked to related autosomal dominant syndromes with limb and brain defects

[Greig Cephalopolysyndactyly, Pallister-Hall, Postaxial Polydactyly]

Answer 13

GLI3 —> loss of Gli3R (repressing Gli —> GOF mutation in Shh pathway)

Question 14

what mutation occurs in the Hh pathway in medulloblastoma?

Answer 14

medulloblastoma: tumor in cerebellum during development

Ptc1 mutation (LOF) causes constant disinhibition of Smo, even in absence of Hh ligand (target genes constantly activated)

Question 15

why is Smo a good drug target and how can it be utilized

Answer 15

Smo is 7-TMR GPCR (most drugs target TMRs)

natural target is NOT Hh ligand

agonists of Smo —> treat LOF Hh diseases

antagonists of Smo —> treat GOF Hh diseases

Question 16

Describe these components of the Wnt signaling pathway:
a. Wnt proteins
b. Frizzled/LRP
c. Axin/APC/GSK3
d. beta-catenin
e. TCF/LEF proteins

Answer 16

a. Wnt proteins: secreted proteins in embryos and adults
b. Frizzled/LRP: co-receptors, bind Wnt
c. Axin/APC/GSK3: destruction complex, mediates phosphorylation of beta-catering for degradation
d. beta-catenin: cytoplasmic protein, controls TCF/LEF activity
e. TCF/LEF proteins: TF that activate or repress target genes depending on signaling status and cofactor association

Question 17

describe Wnt pathway when Wnt ligand is present

Answer 17

1. Wnt binds Frizzled/LRP co-receptors, allowing Dsh to disrupt Axin/APC/GSK3 destruction complex
2. beta-catenin phosphorylation is BLOCKED and it accumulates
3. beta-catenin enters nucleus, binds TCF/LEF proteins as co-factor
4. target genes turned on

Question 18

describe Wnt signaling when Wnt is NOT present

Answer 18

1. destruction complex (Axin/APC/GSK3) phosphorylates beta-catenin
2. beta-catenin is targeted for degradation in proteosome
3. TCF transcription factors in nucleus INHIBIT target genes with co-factor Gro proteins

Question 19

what would you expect from changes in beta-catenin protein stability, localization, or transcriptional activity?

Answer 19

mutations in beta-catenin that prevent phosphorylation or promote stability result in constitutively active Wnt signaling pathway

example: APC mutations in FAP (familial adenomatous polyposis, colorectal cancer)
[APC is part of destruction complex]

Question 20

Which of the following occurs when Hh protein binds to its receptor?
a. Ptc activity is stimulated
b. cholesterol is recruited as a receptor co-factor
c. Smo protein translocates into the cilium
d. IFT proteins are not longer active in signaling

Answer 20

Hh binds receptor —> Smo protein translocates into the cilium

Hh signaling relives Smo and allows it to enter the cilium