Lecture 10- TGFbeta and FGF signaling Flashcards
How are cell fates progressively restricted?
Cells need to communicate with each other during development to instruct a signal to change their behaviour. Change in behaviour often leads to changes in cell fate
Outline the key features of signal transduction pathways
- Reception: the ligand (growth factor, signalling molecule) binds to a cell surface receptor and activates it
- Transduction: receptor activation induces the transduction of the signal from the membrane to the nucleus via a cascade of secondary messenger activation in the cytoplasm
- Response: a transcription factor is activated and induces the transcription of specific target genes within the nucleus
What are the 3 families within the TGF-beta super family?
- BMP-like family
- GDNFs family
- TGF-beta like family
What proteins are classified in the BMP-like family and what are their main roles?
BMPs, GDFs and AMH
- Role in bone formation
- BMPs are important in embryonic development
What are BMP2/4 important for during embryonic development?
BMP2/4 are involved in he specification of the limb and patterning of the mesoderm
What proteins are classified in the GDNF family?
GDNF, Artemin, Neuturin and Perserin
What proteins are classified in the TGF-beta like family and what are their main roles?
TGF-beta, Activins and Nodal
- TGF-beta: important in controlling the formation of the ECM and in cell division
- Activin/Nodal: role in differentiation of the mesoderm, the determination of the A/P axis and the establishment of asymmetry
Describe the TGF-beta signal transduction pathway
- TGF-β ligand binds to TGF-βR2 on the cell membrane
- This triggers the dimerization of TGF-βR2 to TGF-βR1
- When the two receptors become close to each other, TGF-βR2 phosphorylates TGF-βR1
- This phosphorylation activates the receptor
- Activation of the receptor leads to the phosphorylation of an intracellular SMAD complex
- This regulatory SMAD complex enter the nucleus and can either activate or repress target genes and lead to TGF-β-induced cell responses
Describe the BMP signal transduction pathway
- BMP dimer binds to a receptor molecule in the cell membrane
- The ligand binding to the receptor causes subunit II to phosphorylate receptor subunit 1
- Receptor subunit 1 then phosphorylates intracellular Smad1/5 protein
- The phosphorylated Smad protein binds to Smad4 to form a transcriptional regulatory complex
- The regulatory complex enters the nucleus and then either activates or represses target genes. Sometimes transcriptional cofactors are also involved
What is the name given to signal transduction pathways that don’t involve SMAD?
Non-conical pathways
Name 2 BMP antagonists
- Noggin
2. Chordin
How is BMP signalling controlled through inhibitions?
- The antagonists act by binding to the ligand and preventing the BMP from binding to its receptor
- The inhibitors are present at various very restricted places in specific groups of cells in specific tissues
- Through their diffusion, they control the level of response to BMP signalling that occurs within cells
- They play an important role in fine tuning the level of response to BMP signalling that occurs within a cell which is essential in cell fate choice and cell fate specification
Give an example of when BMP inhibitors are used in developing
Involved in the shaping and dorsalising of the mesoderm
How was noggin discovered?
Injected mRNA into artificially ventralised frog embryos and looked at the ability of mRNA to rescue the dorsal structures normally present
What are enzyme linked receptors?
Are transmembrane receptor where binding of an extracellular ligand (often growth factors) causes enzymatic activity on the intracellular side
What are they essential roles enzyme linked receptors are involved in?
Roles in the ways cells proliferate, differentiate and move inside the animal tissues
Why are growth factor responses often slow?
Because intracellularly secondary messengers are numerous and this class of proteins tend to activate numerous second messengers via a cascade
What do growth factors using associate with?
Enzyme linked receptors within extracellular environments as they are not diffusable
Give an example of a class of enzyme linked receptors
RTKs
How many families of RTKs are there and how many RTK genes have been identified within the human genome?
- 20 families of RTKs
* 58 RTK genes identified in the human genomes
Describe the structural features of RTKs
- Mostly monomers
- Extracellular domains vary greatly however there are a few units conserved and repeated (ligand binding activity)
- Intracellular domains are highly conserved and have a catalytic domain which carries kinase activity (enzyme-linked receptors)
- Single transmembrane domain also conserved (25-38 AA)
Which RTK is a dimer?
Insulin receptor
Outline how RTKs are canonically activated
- Activation occurs when the ligand binds to the extracellular ligand binding domain of 2 inactive RTK
- Causes a dimer to form from 2 monomers and they are brought closer together
- The change in conformation activates the intracellular tyrosine kinase domain
- Once positioned correctly, the kinase domains phosphorylate each other (cross-phosphorylation)
- This increases the activity of the kinase, stabilises the receptor in the active state, causes the kinase to phosphorylate other tyrosine residues in the receptor to create Sh2 domain docking sites for intracellular molecules
- The docking sites created in the different receptors will be specific for the intracellular substrates and ultimately leads to the diverse responses in RTKs
What is oligomerisation?
The formation of a dimer from 2 monomers
What type of experimental techniques can be used to analyse RTK signalling?
Loss/gain of function studies
Outline a loss of function study which analyses the importance of dimerisation form RTK signalling
- Using genetic engineering we can generate DNA that encodes for a normal receptor that is mutated in the kinase domain
- This doesn’t affect the dimerization of the 2 tyrosine kinase domains but prevents cross-phosphorylation
- Therefore, ligand binding could still occur but there would be no downstream signalling as the receptor is not activated
- This DNA is then expressed in an organism at high levels and poisons the endogenous receptor (dominant negative)
Outline a gain of function study which analyses the importance of dimerisation form RTK signalling
- Modify the extracellular domain of the receptor to mimic ligand binding
- In the absence of ligand, the receptor would act as if the ligand was present and be constantly activated
How does the phosphorylated RTK transduce a signal?
Phosphorylated tyrosines act as docking sites that can be recognised for various cytoplasmic proteins responsible for mediating downstream signalling
Give 3 examples of pertains that bind to activated RTK and the pathways they are involved in?
- PI3-kinase (inositol lipid pathway)
- GTPase activating protein (RAS/MAPK pathway)
- PLC-gamma (inositol lipid pathway)
What 2 things characterise proteins that bind to activated RTKs?
The SH2 and SH3 domain
What is role of the SH2 and SH3 domains?
SH2 domain in Src recognises this short phosphopeptide
SH3 domain mediates protein interaction and recognised sequences reached
How is Ras converted from an inactive to active state?
GEFs exchange GDP to GTP so is converted into an active state
How is Ras converted from an active to inactive state?
GAPs remove a phosphate from GTP to form GDP, converting Ras into an inactive state
What is bound to Ras in its active and inactive state?
Ras is active when bound to GTP but inactive when bound to GDP
Outline how docking leads to signal transduction, using the Ras pathway as an example
- The phosphorylated RTK receptor now contains docking sites for intracellular proteins (e.g GRB2)
- GRB2 binds through its SH3 domain to Sos which then recruits inactive Ras
- Binding of GRB2 and Sos (GEF) couples receptors to inactivate Ras
- Sos promotes dissociation of GDP from Ras; GTP binds Ras and then it dissociates from Sos
- This leads to the activation of Ras
How is MAPK activated and why is it unlikely to be accidental?
MAPK must be phosphorylated on two residues: a serine and threonine
Dual phosphorylation means it is unlikely to be accidentally activated
Describe the signal transduction downstream of RTKs
- MAPK is activated by phosphorylation by Mek/MAPKK
- MEK/MAPKK uses ATP to provide energy and phosphorylate Serine and Threonine on MAPK
- MEK/MAPKK itself is maintained in an inactive state unless it is phosphorylated
- The protein that phosphorylates MEK/MAPKK is Raf
- Raf can only be activated through binding of activated Ras
- Activated MAPK will then act by activating downstream effectors through phosphorylation (by either directly phosphorylating and activating transcription factors which will lead to activation of transcription of target genes OR phosphorylate other secondary messengers and lead to longer lasting response. These secondary messengers then ultimately activate other transcription factors)
Activated MAPK will then act by activating downstream effectors through phosphorylation by either?
- Directly phosphorylating and activating transcription factors which will lead to activation of transcription of target genes OR
- Phosphorylate other secondary messengers and lead to longer lasting response. These secondary messengers then ultimately activate other transcription factors)
Describe the 3 types of FGF signalling
- Paracrine FGFs: secreted outside the cells and act on neighbouring cells
- Intracrine FGFs: ligands that bind to receptors intracellularly
- Endocrine FGFs: ligands that act long distance by entering the blood stream
Describe the structural characteristics of FGF receptors
- All have an extracellular domain made of D1, D2 and D3 Ig-like domains
- D1 and D2 are separated by an acid box domain whose role is in repressing the activation of the receptor in the absence of the ligand
- Ligands bind to D2 and D3 (provides ligand specificity)
- D2 contains the heparin binding site and have a short transmembrane domain and the kinase domain is split into 2
How many FGF receptors are there and how do their structures compare?
There are 4 FGF receptors which all have a similar structure
What must FGFs be associated with to bind and activate FGF receptors?
Complex with HSPGs
What are HSPGs?
Large molecules that are either associated or bound to the membrane
Why type of FGF ligand have a high affinity for HSPGs and what is the impact of this?
Only paracrine FGF ligands have a high affinity for HSPGs
Paracrine FGF molecules are usually activated at a short distance from the cell because the diffusion cannot occur due to the binding with HSPGs
What is the effect of endocrine FGFs having a low affinity for HSPGs?
Allows them to be secreted outside the cell, diffuse away and enter the blood stream to act at a long distance
What are the 3 different types of HSPG cores?
- Transmembrane
- Tethered
- Secreted
What are the long sugar chains on HSPGs called?
Heparan
How are HSPGs modified and what is the consequence of this?
Each sugar can be modified in many different ways especially by sulphation
Modification could result in a ‘code’ that creates binding sites for specific proteins (e.g FGF2) and lead to the activation of specific receptors
Give 3 examples of pathways FGF signalling can trigger and the processes they effect
- Ras/MAPK pathway: cell proliferation
- PI3/AKT pathway: cell survival
- PLC-gamma pathway: cell motility
Give some examples of diseases associated with FGFR mutations
- Achondroplasia leads to dwarfism
- Pfeiffer syndrome
- Apert syndrome
- Crouzon syndrome
- Thanatophoric dysplasia
- Jackson-Weiss syndrome
