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It can control several distinct and seemingly unrelated phenotypic effects These include 1. Growth control 2. Arrest and proliferation 3. Differentiation
These ligands –
– – – – Are cytokines encoded by 42 open reading frames in humans characterised by six conserved cysteine residues consists of two sub-families (TGFβ /activin subfamily and the BMP (bone morphogenetic protein) subfamily) There are 5 TGFβ ligands (TGFβ 1, 2 and 3 expressed in mammalian cells)
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Active form of TGFβ is a dimer (i.e. 2 units) stabilised by hydrophobic interactions Strengthened by disulphide bonding between each unit. Each monomer comprises extended β -sheets interlocked by 3 conserved disulphide bonds These form a structure known as the “cysteine knot” Dimers suggest a complex with two of each type of receptor
TGF b Receptors •
• • • • • • Receptors are protein kinases that phosphorylate serine/threonine kinases 2 types of receptors : I and II Type II receptors phosphorylate the Type I receptor comprises 12 members All 12 members are dedicated to TGFβ /BMP signalling 1. 7 Type I receptors 3. C-terminal intracellular 2. 5 Type II receptors serine/threonine kinase Each receptor consists of approx domain 500 amino acids • binding of ligand to Type I and 1. N-terminal extracellular Type II receptors initiate signalling ligand binding domain • Signal is then transduced through 2. A transmembrane region Smad proteins
Type I Receptors
• • • • • • Contain GS Domain (TTSGSGSG sequence) GS domain is phosphorylated by the Type II receptor Therefore need both type I and II for active signalling Extensions/truncations to N- and C-termini do exist .
• • There are 3 functional classes of Smad proteins 1. Receptor-regulated Smad Proteins 3. Inhibitory Smad proteins (I-Smad; (R-Smads; Smads 1, 2, 3, 5 and 8) Smads 6 and 7) 2. Co-mediator Smad protein (CoSmad; Smad4))
Mechanism of Ligand Binding There are two distinct modes of ligand binding to the receptors. • • BMPs
1. BMP ligands have a very high affinity for the extra-cellular ligand binding domain of the type I receptors. 2. Once bound the type I receptorligand complex has a a higher affinity for binding to type II receptors than the ligand alone. 3. Receptor-ligand interactions are predominantly hydrophobic and involve a highly conserved phenylalanine residue in the receptor 1. They have a high affinity for type II receptors and 2. They do not interact directly with type I receptors on their own. 3. They bind tightly to the type II receptor domain first. 4. This binding then allows the subsequent incorporation of the type I receptor to form a large ligand-receptor complex. 5. This involves ligand dimers and 4 receptor molecules. 6. Receptor-ligand interactions are predominantly hydrophobic and involve a highly conserved phenylalanine residue in the receptor.
Ligand Receptor Interactions
Mechanism of Receptor Activation •
• • • • • • • Proximity of receptors in the complex facilitates the phosphorylation and activation of the type I receptor by the type II receptor. The kinase of the type II receptor phosphorylates multiple serine and threonine residues in the TTSGSGSG sequence of the type I cytoplasmic GS region. GS region serves as an important regulatory domain for TGFβ signalling. Phosphorylated TGFβ -RI binds efficiently to Smad2 has enhanced phosphorylation specificity for the C-terminal serine residues The phosphorylated receptor cannot be recognised by its inhibitors Therefore phosphorylation activates the receptor by switching the GS region from a preferred binding surface for inhibitors into a binding surface for the R-Smad substrates
Regulation of Receptor Activation •
• • • • • • • Access of TGFβ ligands to its receptors is controlled by two classes of molecules with opposite functions. 1. One class act as ligand binding traps These prevent the ligand from accessing the membrane receptors Ligand binding traps occupy the same hydrophobic pocket in the ligand that the receptor requires for binding Therefore they block binding 1. The second class acts as accessory receptors or co-receptors They promote ligand binding to the signalling receptors They have some selectivity in this role TGFβ type III receptor (a proteoglycan) enhances signalling by mediating binding of growth factors to the TGFβ -RII.
Recognition of Smads by Activated Receptors
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TGFβ type III receptor is critical for TGFβ 2 signalling, but the type III receptor does not bind to activin or to BMPs.
R-Smads have a much more positively charged surface next to their L3 loop than CoSmads. This basic patch provides a binding surface for the phosphorylated GS region from the activated receptor. Interaction of the basic surface with the phosphorylated residues enhances the receptor binding affinity Specificity is controlled by the L45 loop of the receptor kinase domain L45 loop of the activated receptor interacts with the L3 loop in the Smad Matching sets of L45 and L3 loops will determine Receptor-Smad interactions
Regulation of Smad Access to the Receptors
• • • • • Recognition of R-Smads may be facilitated by auxiliary proteins. Smads 2 or 3 immobilised Smad Anchor for Receptor Activation (SARA). Bind through interactions between a peptide sequence of SARA and an extended hydrophobic surface on Smads Targeted to the membrane of early endosomes. The activated receptor-ligand complex is internalised via clathrin coated pits into the early endosomes where the SARA-bound Smad can be activated.
Mechanism of Smad Phosphorylation and Activation
• • •
Phosphorylation of R-Smads by the kinase of the type I receptor occurs on the terminal serine residues in the flexible SXS motif Phosphorylation 1. destabilises the complex with SARA 2. allows dissociation of the Smad from the complex 3. exposes a nuclear import region increases its affinity for the Co-Smad, Smad4. 4. The formation of the R-Smad-Co-Smad complex brings about the assembly of the transcriptional regulation complexes The terminal end of one of the β -strands moves in response to phosphorylation This weakens the Smad interaction with SARA Results in decreased binding affinity as this β -strand is required for the Smad-SARA interaction
Growth Control • Inhibition of cell proliferation: inhibits the cell cycle during the G1 phase. by inhibition of the (CDKs) and down regulation of c-myc. Cell Death • TGFβ can induce Apoptosis in various tissues and organs. • lactation : a rise in TGFβ3 levels mediates the apoptosis that precedes mammary gland involution. • TGFβ and Cancer 1. cancer Inhibitor • all pancreatic and colon cancers have mutations in TGFβ signalling pathway • This suggests a role for TGFβ in tumour suppression.
Cancer Promoter • TGFβ expression correlates with advanced clinical stage of tumours. • TGFβ can contribute to tumour growth by – suppressing the immune system or – stimulating the production of angiogenic factors • TGFβ can also act directly on the tumour cells – cells may exhibit enhanced migration – invasive behaviour in response to TGFβ . – TGFβ can induce an de-differentiation of tumour cells in an epithelial to mesenchymal transition (EMT). • EMT is characterised by – down regulation of the proteins involved in cell-cell adhesion and – up-regulation of the proteins that are important for ECM association – This leads to increased migratory and invasive potential. TGFβ Receptor Mutations in Cancer
Inactivating mutations in the TGFβ -RII occur in most colorectal and gastric cancers with micro-satellite instability (MSI). • MSI is a common occurrence in many sporadic cancers. • Mutations are uncommon in MSI tumours from endometrial, pancreatic and liver cancers. • TGF-RII loss is selected for only in tumours of specific tumour origins. • Mutation of the TGF-R1 has also been detected in human cancers. Smad Mutations in Cancer • TGFβ signalling network is also disrupted by mutations in Smad2 and Smad4. • Smad4 mutations occur in half of all pancreatic cancers and one third of colon cancers. • Mis-sense mutations usually result in a loss of stability of molecules and disrupt formation of the trimers that are required for signalling. BPH • BPH – Benign Prostatic Hyperplasia • Age related enlargement of the prostate • Time of life anomaly with estrogen:androgen ratios changing • Benign non-malignant condition • Symptoms include alterations in ability to urinate (incomplete voiding and more frequent urination) • Characterised by an increase in the epithelial:stromal ratio from 1:2 to 1:5 • Evidence that TGFβ was involved so research aimed to investigate this hypothesis
BPH Theory • Low levels of TGFβ stimulate cell proliferation • Estrogens go up • More TGFβ More proliferation,Growth arrest,Cell differentiation • Lots of ECM produced • Stromal compartment of the prostate gets bigger Diabetic Nephropathy • Diabetes is characterized by absolute or relative insulin deficiency that leads to hyperglycaemia and an altered glucose, fat, and protein metabolism • Hyperglycemia is a most important factor in the progression of diabetic nephropathy • Early alterations in diabetic nephropathy include glomerular hyperfiltration, glomerular and tubular epithelial hypertrophy, and the development of microalbuminuria • TGF-β has been recognized as a profibrotic growth factor • involved in the expansion of mesangial matrix • Elevated levels of TGF-β have been measured in the glomeruli of diabetic rats • Neutralizing TGF-β antibodies prevented diabetic renal atrophy, mesangial matrix expansion • Certain TGF-β -inducible genes appear to exert fibrogenic effects on diabetic kidneys • Serum and urinary TGF-β levels have been found to correlate with the severity of microalbuminuria.
AGE = Advanced glycation end products; MCP-1 = monocyte chemoattractant protein-1; ECM = extracellular matrix; GBM= glomerular basement membrane; PKC =protein kinase
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