Lecture 14 Flashcards
Explain the TGF-? signal transduction pathway following ligand binding
The activated Type I receptor phosphorylates Smad proteins. The type of TGF-? ligand that binds to the receptor determines the specific smads phosphorylated by the activated receptor. BMP ligand binding leads to phosphorylation of Smad1,5 and 8 whilst TGF-? binding results in Smad2 and 3 phosphorylation. Smads1,2,3,5 and 8 are collectively known as receptor-regulated or R-Smads. R-Smads are maintained near the plasma membrane close the kinase domains of the receptors. When the pathway is inactive the R-Smads are anchored at the cells membrane by Smad anchor for receptor activation proteins (SARA). Once R-Smads have been phosphorylated by the activated Type I receptor they oligomerise with Smad4 to form heterodimers. This Smad complex then migrates to the nucleus where its binds to the DNA to regulate gene expression
What kind of bonding hols the LAP and TGF-? dimers together in a complex
Disulphide bridges between adjacent cysteine residues
Explain the processes that lead to the release of the active ligand from the complex
Release of the TGF-?/LAP/LTBP complex from the extracellular matrix involves cleaves by the proteases plasmin and calpain that releases the complex from the extracellular matrix. Subsequent binding of thrombospondin to LAP releases the active TGF-? ligand
Which TGF-? ligand family have an important role in left/right patterning and splitting of bilateral symmetry
Nodal
What does TGF-? stand for
Transforming growth factor-?
How can Smads lead to a decrease in gene transcription
Smads can also bind to co-repressors and recruit histone deacetylases (HDACs) which remove acetyl groups from the histones and decrease gene transcription by tightening the chromatin structure.
The BMP family of TGF-? ligands are negatively inhibited by several different proteins that act as extracellular antagonists. List 5 of these such BMP antagonists
Noggin, Chordin, Follistatin, Cerberus, Gremlin
What type of mutations related to TGF-? signalling are often found in patients with certain cancers
Hypomorphic (partial loss of function) mutations
What are the four ligand subfamilies of TGF-? signalling
Activin, Nodal, BMPs/GDFs and TGF-?
The TGF-? precursor is cleaved into two parts by the action of a protease, what are these two regions and what is their role
The C-terminal region is responsible for the formation of the active ligand by dimerisation with other TGF-?s. The N-terminal part binds to TGF-? and is called Latency Associated Protein (LAP)
Decoy receptors can also inhibit TGF-? signalling, give an example of this
The BAMBI pseudo-receptor resembles the Type I receptor but lacks the intracellular kinase domain. It forms an inactive complex with the ligand and the Type II receptor and inactivates BMP, activins and TGF-? signalling
Structurally, Type I and Type II TGF-? receptors are very similar. What action do they both have
They are both serine-threonine kinases but have different functions in the pathway
How many classes are there of general TGF-? antagonists and what is the purpose of these different families
There are five different families of TGF-? negative regulators these allow for distinct expression profiles allowing for greater refinement of the signal by binding of different subsets with different affinities.
What is significant about Smad4
Smad4 is constitituvely expressed and unlike the other Smads, it is common to all TGF-? signalling pathways. It is therefore referred to as the common-mediator or Co-Smad
Lefty and antivin are TGF-? antagonists that inhibit Nodal signalling, explain how these pseudo-ligands act
These antagonists bind to the Type II receptors but lack an ?-helix loop regions that allows for dimerisation and the cysteine resides responsible for disulphide bridge formation. This ultimately results in the inability of the Type II receptor to bind to the Type I receptor
What is the role of Latent TGF-? binding protein (LTBP)
LTBP tethers the LAP part of the complex to components of the extracellular matrix to increase the effective concentration of the ligand by restricting its diffusion
What are the two ways in which TGF-? signalling antagonists can act
They can either compete for binding at one site on the ligand (I.e. gremlin, noggin, cerberus and DAN on BMP2) or, antagonists can bind to independent sites allowing for multiple inhibitor binding
Which TGF-? ligand family have an important role in hormonal regulation
Activins
What are the three key domains of an R-Smad and what is their function
The MH1 domain in the amino terminus is responsible for binding to the DNA. There is an MH2 interaction domain that controls protein-protein interactions and regulates the homo/heterodimerisation of Smads. Finally, R-Smads also contain a domain in the carboxyl terminus that acts as a recognition site for phosphorylation by the activated Type I receptor
Describe the activation of the TGF-? receptor following ligand binding
Activation of the receptors comes from the formation of a dimer receptor complex. The Type II TGF-? receptor binds to the TGF-? ligand homodimer and then recruits and phosphorylates the Type I receptor. This leads to the phosphorylation of Smads and further downstream signal transduction
What are the results of mutations in Smad4
Disruption of all TGF-? signalling pathways
How does the structure of an I-Smad differ from that of an R-Smad
Whilst I-Smads have retained the MH2 interaction domain they lack the functional MH1 DNA binding domain as well as the phosphorylation domain
Explain how both hypomorphic and amorphic TGF-? signalling mutations impact cancer and tumorigenesis
Null/apomorphic mutations cause benign lesions which cannot go through EMT. Hypomorphic mutations increases rate of benign lesions, by decreasing inhibition of clonal expansion, with enough signalling still conserved to trigger later stage EMT and metastasis.
Give an overview of the TGF-? signalling pathway
The TGF-? ligand binds to the extracellular domain of the Type II TGF-? receptor and promotes its further binding to the Type I TGF-? receptor via the ligand also. The activated TGF-? receptor dimer undergoes transphosphorylation of the Type I receptor by the action of the intracellular serine-threonine kinase domain of the Type II receptor. This transphosphorylation causes the binding of a cytosolic Smad proteins which then get phosphorylated, dimerise with other Smads and translocate to the nucleus where they regulate gene transcription.
