TGF-B and FGF Signalling Flashcards
What is Paracrine Signalling?
Extracellular signals where ligands diffuse through extracellular space and go to local cells to bind and signal
What are common features in signal transduction pathways?
Reception: Ligand binds to cell surface receptor and activates it
Transduction: Receptor activation activates cascade of second messengers which transmit message from membrane to nucleus
Response: Transcription factor is activated and induces the transcription of specific target genes
What does the TGF-B superfamily consists of?
30 dimeric ligands (two subunits joined together)
What are the two subfamilies within the TGF-B superfamily?
- TGF-B-Like family: includes the TGF-B themselves and often used in cell proliferation and symmetry
- BMP-like family: includes BMPs themselves (and GDFs and AMHs)
What is the distinction between the two families in the way that they signal?
TGF-B = Uses smad 2/3
BMP-like = Uses Smads 1/5/8
**Both use Smad4
Explain how Smad Signalling occurs
- TGF-B ligands binf to Type II TGF-B receptor dimers
- Subsequent recruiting and phosphorylation of type 1 TGF-B receptor
- Type 1 receptor phosphorylates cytoplasmic tail of Smad proteins (2/3)
- Once phosphorylated, Smad 2/3 form complex with Smad4 and the complexes are translocated to the nucleus
- Interaction with TFs
What are the TGF-B receptors?
Serine-threonine kinases
Explain how BMP signalling works
- BMP ligands bind to BMP type II receptors
- Subsequent recruiting and phosphorylation of BMP type 1 receptors
- BMP type 1 receptors phosphorylate receptor-regulates Smad proteins (1,5,8)
- They form complex with Smad 4
- Phosphorylated complexes translocate into the nucleus
How can immunoflouresence be used to detect BMP signalling?
- Visualise phosphorylated Smad detection which shows activated BMP signalling
- Detect chordin and noggin to see where Smad phosphorylation hasn’t happened
How can we use genetic engineering to look at functions of TGF-B?
- Generate DNA that encodes a mutated receptor
- E.g. one that is constitutively active/dead
- Can see the effects of ectopic BMP signalling as it is expressed in cells/embryo in diff. place
Give an example in zebrafish of how we can interfere with the pathway to see its effects
- Smad 2/3 signalling is required for mesoderm formation
- Nodal ligands stimulate this signalling in vivo
- oep = mutant for cripto which is an essential Nodal co-receptor
- Can see the effects of mesoderm formation
Give some properties of receptor tyrosine kinases
- Normal mediate signals from ligand growth factors
- These growth factors act in a paracrine fashion
- Some ligands can bind to many receptors and vice versa (some receptors can have many ligands that bind)
- Most tk are monomers
- Variable extracellular domain where ligand binds and conserved intracellular domain where tyrosine activity occurs
How does Signal activation occur at tyrosine kinase receptors?
- Ligands will dimerise for receptor binding
- Dimerised ligand binds to two receptors
- Once positioned, the kinase domains cross-phosphorylate each other as they both have a tyrosine kinase domain
-Increases kinase activity
How is kinase activity increased after dimerization has occurred?
- Can phosphorylate more proteins
- Can increase receptor stability
- Can cause phosphorylation of other tyrosines in the receptor to create docking sites
How do proteins recognise phosphorylated docking sites?
Via SH2 domain which has phospho-tyrosine domain which recognises the phosphorylation sites in the receptor
- The proteins with an SH2 domain also have SH3 domains which interact with the other proteins (Ras Pathway)
Explain how the Ras pathway works
Ligand binds to transmembrane receptor and pulls two together
- Phosphorylation of tyrosine happens leaving a phosphorylated receptor ligand complex
- Grb2 then binds it has a SH2 domain which recognises phosphorylated tyrosine residues
- SOS (Guanosine nucleotide exchange factor ) binds to Grb2
- SOS helps RAS (G-protein) exchange its GDP for GTP (activating RAS protein)
- RAS interacts with RAF and then MEK, phosphorylating it and activating it
- MEK then activates and phosphorylates ERK which then translocates into the nucleus
- Phosphorylated ERK interacts with transcription factors allowing gene transcription to happen
- The genes transcribed are genes that control cell growth and division (proliferation)
How does the MapK pathway work and what is it important for?
MapK essential for proliferation, differentiation etc.
1. Activated Ras activates Map3K
2. Activated Map3K phosphorylates MapK2, activating it
3. Map2K activates MapK by phosphorylating it
4. Activated MAPKs then regulate activity of various transcription factors and other proteins
What are some of the families FGF ligands are split into?
22 ligand members split into:
- Paracrine family
- Endocrine family
- Intracrine family
What are the 4 receptors all FGFs signal throuhg?
FGFR1, FGFR2, FGFR3, FGFR4
- Gene expression of the different ligands is what causes its differential binding/cascade
Explain the structure of FGF receptors
- 3 immunoglobulin like domains (D1, D2, D3)
- Ligands bind to D2 + D3
- Has a transmembrane domain
- Has a kinase domain split into two parts
What is the role of HSPGs in FGF receptors?
- Activation of FGFRs requires HSPGs (long sugar chains modified by sulphation)
- Determinant of binding
- HSPGs have a protein core that can be transmembrane, tethered, or secreted
- HSPGs specify a ‘code’ for what ligand can bind
- FGF can only activate its FGFR if its in a complex with the HSPG
What is the difference between paracrine and endocrine FGFs and their affinity for HSPGs?
- Paracrine FGFs have high affinity for the HSPGS, means they act locally and stay near from where they are secreted from
- Endocrine FGFs have a low affinity for HSPGs so they diffuse into blood stream
Give some examples of the second messenger cascades that FGF activation can lead to
- MapK pathway for proliferation
- AKT pathway for cell survival
Depends on which protein docks to the phosphorylated sites
How are FGFR mutations associated with disease?
- Different proteins within the second messenger mutations can lead to different diseases
- Achondroplasia (type of dwarfism)
- Apert syndrome
