1. In what subclasses can you divide the FGF ligands and what are the characteristic of each subclass regarding structure and functional properties?
All have the conserved CORE with about 120 aa
Intracrine FGFs (FGF11-14)
Lack the N-terminal signal sequence for secretion = signal peptide [SP] (-> signal inside a cell)
Have the heparin binding domain (HB)
Paracrine FGFs
have SP -> secreted but also the HB -> tightly bind to heparan sulfate proteoglycans of the extracellular matrix (ECM)
signal in an area close to the cell (local)
Endocrine FGFs (FGF15/19, FGF21, FGF23)
have SP -> secreted and no HB -> diffuse over long distance
they can enter bloodstream
Summary 1: The FGF Family
• The FGF family in chick, mouse, and human consists of 22 structurally related proteins.
• FGFs can be divided into three classes: intracrine, paracrine, and endocrine FGFs.
• Paracrine FGFs can further be divided into 5 subfamilies based on phylogenetic analysis.
• Intracrine FGFs (FGF11-14) contain a heparin-binding domain, but no signaling peptide and are not secreted.
• Paracrine FGFs contain a signal peptide and heparin-binding domain, and are secreted and act locally.
• Endorine FGFs contain a signal peptide but lack a heparinbinding domain and are secreted, enter the blood stream and act at long distances.
• During evolution, the FGF family developed through several rounds of gene duplications form an ancestral gene already present before the diversion of protostomes and deuterostomes.
2. What changes does FGF binding to its receptor trigger leading to receptor activation?
Dimerization upon Ligand binding (Protein-Protein Interaction)
FGFs with HB -> crosslink by HSPG: Heparan sulfate proteoglycan (different specificity for HB FGFs)
both leading to conformational change inside where the tyrosine kinases TRANSPHOSPHORYLATE each other = receptor activation
Endocrine FGFs use Klotho proteins (a/ß) as Co-receptors
3. FGF receptors are alternatively spliced: How is this process regulated and what is the consequence of this alternative splicing?
How?
tissue specific RNA binding proteins
intronic or exonic splicing enhancer (ESE, ISE) or silencer sequences (ESS, ISS)
Consequence
4 FGF receptors mediate the effects of 18 secreted FGF ligands
Ligand-binding specificity is regulated by alternative splicing
e.g. Epithelial tissues predominantly express the IIIb isoform (FGF10) and mesenchymal tissues express IIIc (FGF1)
Summary 2: The FGF receptors and co-receptors
• Humans, mice and birds have 4 Fgf receptor genes (Fgfr1-4) encoding high-affinity transmembrane tyrosine kinase receptors.
• Each FGFR receptor is capable of binding to different FGFs; similarly, each FGF is capable of binding to different FGFRs.
• FGFRs consist of 3 extracellular immunoglobulin (Ig) domains, a single transmembrane helix and an intracellular split tyrosine kinase (TK) domain.
• The second and third Ig domains form the ligand-binding pocket and have distinct domains that bind both FGFs and HSPGs.
• For FGFR1-3, ligand-binding specificity is regulated by alternative splicing of the Ig III domain giving rise to IIIb and IIIc isoforms predominantly found in epithelial and mesenchymal tissues, respectively.
• HSPGs are essential for the assembly of the FGF-FGFR complex for paracrine FGFs
• Endocrine FGFs use Klotho proteins as co-receptors.
4. How are activated FGF receptors recognized by other proteins?
intermolecular transphosphorylation of the tyrosine kinase domains and intracellular tail —> Phosphorylated tyrosine residues on the receptor function as docking sites for adaptor proteins
Recognized via:
SH2 Src Homology 2 Domain
PTD Protein transduction domain
5. How do you analyze in vitro, which FGF ligand binds to which receptor variant? Is there anything problematic with this kind of experiments? Can you expect that the results also apply to the in vivo situation?
e.g. cell line that expresses a specific receptor but has no FGF production, then add each FGF ligand and analyze which one has an effect
(mitogenic effect -> observe amount of proliferation equals strenghth of binding )
Problem in vitro: no HSPG —> important interaction missing for many FGFs thats why in that case the in vivo situation could be different
Also endocrine FGFs spread over bloodstream in vivo that might be diffent to a cell culture too
6. What transcription factors are activated downstream of the FGF signaling pathway?
Ets proteins, AP1, GATA proteins, c-myc, and CREB
ELK1 (ETS-like gene 1):
A downstream target of the MAPK/ERK pathway, ELK1 is involved in the regulation of genes associated with cell proliferation and differentiation.
FOS and JUN (AP-1 complex):
The FOS and JUN proteins form the AP-1 (Activator Protein-1) transcription factor complex, which is activated by the MAPK/ERK pathway. AP-1 regulates genes involved in cell growth, differentiation, and apoptosis
MYC:
The MYC oncogene (Ras-MAPK pathway) involved in cell cycle progression and growth.
7. Which negative feedback inhibitors of the FGF pathway do you know, and how do they act?
Sef, MKP1&3, and Sproutys
SEF: inhibit the FGFR directy or phosphorylation and activation of ERK
SPRY1-4: inhibit the activation of Ras by binding to the adaptor protein Grb2 or the guanine nucleotide exchange factor Sos
MKP3: phosphatases that dephosphorylate and inactivate MAPKs
Summary 3: Intracellular signal transduction
• FGF binding to its FGF receptor induces receptor dimerization and a conformational shift in receptor structure that activates the intracellular kinase domain, resulting in intermolecular transphosphorylation of the tyrosine kinase domains and intracellular tail.
• Phosphorylated tyrosine residues function as docking sites for adaptor proteins which activate several intracellular pathway.
• The most important pathways activated by activated FGF receptors are the Ras/MAPK/ERK pathway, the PI3 kinase/Akt pathway and the the PLCg/Ca2+ pathway.
• Downstream, diverse cellular processes including cell proliferation, cell survival, cell adhesion and cell shape, cell migration and cell differentiation are regulated.
8. How would you analyze, in which cells of an embryo FGF signaling is activated?
study function of FGFs by knocking it down (disrupting it) in embryos by understanding its function it is easier to analyze where it is activated
Use an antibody staining specific to phosphorylated kinases so only signal where active FGF downstream kinases
Use Reporter mice with FGF transcription factor regulatory region reporter so whenever FGF active fluoresence proteins are expressed
in situ hybridisation of FGF target genes - possible crosstalk with other pathways
9. Why are the functions of FGF signaling in the adult stage still rather poorly characterized?
Death: Easiest way to find out function is genetic knockdown and most of these die in embryo stage the FGF pathways are just vastly to important in developement!
Also the redundancy, complexity and ability for compensation of the FGF Pathways, very much cross talk, often FGF may also only have a subtle role in adults especially compared to developement
Solution might be conditional knock outs (rekombinases) where the target gene is disrupted only when the adult organsism is treated e.g. with a specific drug or use an inducible promotor that is only activated when treated e.g. with heat shock
10. What changes occur in mouse blastocysts cultured in medium with a high concentration of FGF4 protein compared to blastocysts cultured in a standard medium without additional FGF4?
high —> primitive endoderm (inner cell mass) —> FGF4 activating FGFR2 and the downstream TF GATA6 which reinfoces the FGFR and TF NANOG is inhibited leading to PrE
recent studies also show FGFR1 important during cell fate decicion
without —> epiblast
11. What is the role of FGF8 and FGFR1 during mouse gastrulation?
Studied by using Knockout Mice —> no Epithelial to mesenchymal transition (EMT) after migration towards promitive streak because of E. cadherins that dont release the cells there —> FGF8 activates FGFR1 which activates SNAIL which inhibits E. cadherin
-> disrupted cell fate differentiation
FGF8 is expressed at the primitive streak
FGFR1 function studied by using chimeric mice
maintenance of TBX6 via Brachjury —> Patterning
12. Activating mutations in FGF receptor are involved in human genetic diseases (e.g. skeletal dysplasias) but also in cancer. Explain the difference!
a mutation in a somatic cell can result in cancer (like an hyperactive FGFR that gives oncogenic properties to the cell)
a mutation in a FGF(R) gene in stem cells can be inherited from the parents and cause diseases that affect developement and the whole organism directly
13. Which strategies can be used to inhibit FGF signaling? What are the advantages and disadvantages? Which of those might also be useful for human therapies?
General: chemicals cheaper, better uptake method vs. Antibodies that are very specific
!Stability in the organism, toxicity / side effects, uptake (matabolism)
Small molecule inhibitors:
adv.: oral availibility, reversible (when not taken anymore), cheap, broad usage, potency
disadv.: toxicity / side effects, resistance
a) allosteric receptor binding
b) orthosteric
d) kinase inhibitors —> even more off-target effects in many other signaling pathways (and ana/catabolism) because kinases are everywhere
Monoclonal Antibodies
adv.: very specific
disadv.: cost, intravenous injection, heavy immune response
c) ligand traps
adv.: very specific, but also versitile towards different FGFs
disadv.: complexity, cost, short half life
small molecule inhibitors and monoclonal antibodies are the most advanced and widely used in clinical settings, particularly for treating cancers with aberrant FGF signaling
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