Receptor Tyrosine Kinases Flashcards
RTKs general
58 human RTKs
Examples include cell surface receptors for Epidermal Growth Factor (EGF), Vascular Endothelial Growth Factor (VEGF), Insulin, Fibroblast Growth Factor (FGF), Platelet Derived Growth Factor (PDGF) and Hepatocyte Growth Factor (HGF).
They are key regulators of critical cellular processes such as proliferation, differentiation, cell survival, cell migration and cell-cycle control.
Have a ligand-binding domain in the extracellular region, a single TM α-helix and a cytoplasmic region that contains the protein kinase domain, plus additional C-terminal and juxtamembrane regulatory regions.
Mutations in RTKs and aberrant activation of their intracellular signalling pathways have been causally linked to cancers, diabetes, inflammation, severe bone disorders, arteriosclerosis and angiogenesis.
RTK families
The insulin and EGF receptors show some similarities in their domains. The both contain leucine-rich domains and cysteine-rich domains. However, the insulin receptor is disulphide linked, so it is the only receptor inherently present as a dimer.
MET is often involved along with EGFRs in lung cancer.
VEGF and PDGF receptor families have very similar structures - they only differ in the number of Ig domains, with VEGFRs having 7 and PDGFRs 5. They both have a split kinase intracellular domain.
RTK dimerisation
GF binding activates RTK by inducing dimerisation.
The insulin receptor and insulin-like growth factor 1 (IGF-1) receptor are exceptions, expressed as disulphide linked dimers even at rest.
Monomeric EGF induces receptor dimerisation, but it may also bind and activate pre-existing oligomers.
In the inactive state, the EGFR extracellular domain is folded up and the dimerisation domain (II, Cys-rich) is buried.
EGF binding causes a large conformational change that opens up and exposes the domain, allowing dimerisation, whic leads to autophosphorylation and activation.
Certain RTKs, like Tie2 which responds to angiopoietins, may require the formation of larger oligomers for activation.
RTKs can form homo or hetero-dimers.
HER2 has no known ligand and the dimerization domain is exposed in its resting state, ready to dimerise with any other activated (or resting) EGF family receptor. HER3 has no active kinase domain. These receptors can form heterodimers.
PDGFRs
Platelet-derived growth factor (PDGF) receptors can form both heteromers and homomers.
The ligands are dimeric: PDGF-AA, -BB, -AB, -CC, and –DD.
Ligand binding to PDGFRs maintains and stabilises endothelial tubes during development, promotes endothelial cell proliferation and induces angiogenesis.
PDGFR-β on pericytes is also important for pericyte recruitment during angiogenesis and wound healing to stabilise the blood vessels. PDGFR-β on pericytes interacts with neuropilin-1 co-receptors on endothelial cells. The heteromeric complex can bind PDGF-DD.
The receptors can form α or β homomers, or they can form heterodimers containing one of each subunit.
There is scope for complex signalling depending on the locally-produced growth factors and receptor distribution.
VEGFR subtypes
There are 3 receptor subtypes: VEGFR1 (Flt-1), VEGFR2 (Flk-1/KDR), VEGFR3 (Flt-4).
VEGF ligands are homodimers. Different ligand isoforms bind different receptors to fulfil different functions:
- VEGFR1 binds VEGF-A and VEGF-B. It regulates monocyte and macrophage migration.
- VEGFR2 binds VEGF-A, VEGF-C and VEGF-D. It regulates CV, haematopoietic and lymphatic development and angiogenesis.
- VEGFR3 binds VEGF-C and VEGF-D. It regulates lymphagiogenesis.
VEGF-A is the most important ligand in angiogenesis signalling. These can be alternatively spliced.
Cross RTK family interactions and co-receptors
Cross family interactions are seen between PDGF and VEGF ligands as the receptors are closely related, only differing in the number of extracellular Ig domains.
The VEGF co-receptor neuropilin-1 plays a role in PDGFR-β signalling, seen between pericytes and endothelial cells.
Interactions can be on the same cell (cis conformation) or between two different cells (trans conformation).
Effect of growth factors on extracellular receptor regions
Binding of a NGF dimer crosslinks two Tropomyosin receptor kinase A (TrkA) molecules without direct contact between the receptors. The GF dimer is cysteine-bound, but the receptor proteins are not in direct contact with each other, forming contacts through the ligand instead.
A stem cell factor dimer crosslinks two KIT molecules (PDGFR family). Two Ig domains, D4 and D5, reorientate upon receptor activation, causing the conformational change required for kinase activation.
Two fibroblast growth factor receptors (FGFR) contact each other through their D2 Ig domains. Heparan sulphate proteoglycans (HSPGs) also connect these domains. In addition, each FGF molecule contacts domain D2 and D3 of both receptors.
- Heparan sulphate is a long-chain polysaccharide ubiquitously expressed on all cell surfaces. It can bind to and capture growth factors and deliver them to the receptor.
- The ligand is dimeric
Binding of two monomeric EGF molecules drives conformational changes in the receptor exposing a previously occluded dimerization domain.
Proteoglycans play a role in delivering Hepatocyte Growth Factor to its receptor MET.
Eph/EPHRIN signalling
Eph/EPHRIN signalling occurs across cell-cell junctions.
Erythropoietin-producing human hepatocellular (Eph) proteins constitute the largest known receptor tyrosine kinase family, with 14 members in humans.
The agonist EPHRIN is membrane-bound and can be found on a separate cell to the receptor, not free in solution.
EPHRIN-A is attached by a glycosyl phosphatidylinositol (GPI) anchor
EPHRIN-B is attached by a single transmembrane domain.
The Eph/EPHRIN signalling family has a myriad of roles in development, bone homeostasis, the immune system and cancer.
Activation of intracellular kinase domains
Tyrosine kinases have an N-lobe and a C-lobe.
The key regulatory elements are the activation loop and the N-lobe αC helix that adopt a specific configuration in all activated tyrosine kinase domains.
In the FGFR and insulin receptors, the activation loop interacts directly with the active site of the kinase and blocks access to protein substrates, ATP, or both. Phosphorylation disrupts these autoinhibitory interactions.
In the PDGFR family, including KIT, the juxtamembrane region interacts with elements of the active site to stabilise an inactive conformation. Phosphorylation allows the kinase domain to adopt an active conformation.
The EGFR TKD is allosterically activated by contacts between the C-lobe of the activator TKD and the N-lobe of the receiver TKD across the dimer interface. The activator TKD destabilises auto-inhibitory interactions that involve the activation loop of the receiver TKD.
EGFR signalling pathways and adaptor proteins
EGFR signalling leads to gene transcription and cell proliferation.
Ras and PI3K bind through scaffold proteins. PKC can also directly bind.
Ras mutations are particularly important in cancer development, as they can cause constitutive activity.
The scaffold proteins involved in these cascades have common regions, called SH2 and SH3 regions.
Grb2 is a scaffold protein that has an SH2 domain that finds phosphorylated tyrosine residues and starts to assemble a scaffold of signalling proteins.
It also has an SH3 domain which is bound by proline-rich regions of other signalling proteins, such as SOS.
Within SOS, there is a G-protein, which leads to the activation of Ras.
Protein-protein interactions build up on the active receptor, leading to the signalling cascade.
Different SH2 domains exhibit distinct binding preferences for phosphotyrosine-containing proteins, so the specific interactions between SH2 domains and their ligands plays a crucial role in determining which signalling proteins associate within a cell following growth factor stimulation.
Alpha screen
These screens use donor beads that can be activated by lasers at 680 nm.
If this gets close to an acceptor bead, a free oxygen can move across causing an emission at a lower wavelength (520-620 nm).
One bead can have an antibody that recognises phosphorylated MAPK, and the other one that recognises MAPK. The beads will only come together and produce a signal if MAPK is present and it has been phosphorylated, showing that signalling is active.
Reporter genes
Reporter genes drive the expression of proteins that can be fluorescent (e.g. GFP), bioluminescent (luciferase) or easy to measure in a colour reaction (secreted alkaline phosphatase).
A sequence that recognises one of the transcription factors from one of the signalling pathways is added.
For example, a serine response element (SRE) can be used for the MAPK pathway, and Activator protein-1 (AP-1) or an NFAT-RE for the PLC-𝛾 pathway.
Looking at MAPK signalling, if the signalling system sends a transcription factor onto the SRE, it will drive the expression of the fluorescent protein GFP. This can be easily observed.
AP-1 drives the expression of secreted alkaline phosphatase, which produces a single yellow-brown reaction that allows you to measure how much of the signalling protein you have using colourimetry. This is also very cheap.
VEGF basics
VEGF promotes endothelial cell proliferation, migration, survival and vascular permeability - angiogenesis.
Works in concert with other mediators, such as fibroblast growth factors (FGFs), platelet-derived growth factor (PDGF), matrix metalloproteinases (MMPs), and Notch receptors.
Plays an important physiological process in exercise (repairing microtears), wound healing, menstruation, granulation formation and neuroprotection
Aberrant VEGF signalling is seen in pathophysiology, such as cancer (hypoxia), rheumatoid arthritis, macular degeneration, diabetic retinopathy and infantile haemangioma.
VEGFs are a family of proteins comprising VEGF-A, VEGF-B, VEGF-C, VEGF-D, Placental Growth Factor (PIGF), viral encoded VEGF-E, and VEGF-F (snake venom derived).
These have a common quaternary structure despite low % homology. They are all members of the ‘Cysteine knot superfamily’ of proteins, alongside other proteins such as PDGF and TGF-β.
Biologically active as anti-parallel homo- and occasionally heterodimers (VEGF-A and VEGF-E).
VEGF-A is the best characterised and most potent angiogenic stimulator, secreted by endothelial cells, fibroblasts, smooth muscle cells, platelets, neutrophils, macrophages and ~60% of solid tumours.
Transcription and secretion can be regulated by hypoxia, ischemia and inflammatory mediators.
The gene is upregulated in many tumours and expression correlates with development and metastasis.
VEGF-A structure
VEGF-A is arranged as an antiparallel homodimer.
Each monomer consists of 7 β-strands and 2 ⍺-helices, linked to each other by 3 intramolecular disulphide bonds and to the other monomer by two intermolecular disulphide bonds.
Monomer association is aided by hydrophobic interactions that occur perpendicular to the plane of the β-strands.
VEGF-A isoforms
The Vegfa gene coding region spans ~14 kilobases and consists of 8 exons and 7 introns that can be alternatively spliced to produce >16 isoforms.
These isoforms differ in length, bioavailability and ability to interact with VEGF binding receptors.
VEGF145a seems to be highly expressed in the kidney, a highly vascularised organ
Exons 2, 3 and 4 are responsible for receptor binding - found in all isoforms.
Exons 6a, 6b and 7 are responsible for binding to neuropilin co-receptors and the extracellular matrix. 121 and 111 lack these ECM binding regions and are therefore freely diffusible, going to any site of angiogenic need. Longer isoforms, like VEGF206a and VEGF189a are localised → only cleaved off by MMPs when they are required for angiogenesis, otherwise inactive.
VEGF-165a is the prototypical isoform, containing exons 7a and 8a but lacking exon 6.
We can have proximal or distal splicing at the exon 8a/8b boundary (procimal vs distal splicing site).
- 8a splicing produces VEGFxxxa, such as VEGF165a, VEGF121a and VEGF189a, which are pro-angiogenic proteins → vascular permeability, cell survival, proliferation, migration, angiogenesis
- 8b splicing produces VEGFxxxb, such as VEGF165b, VEGF121b and VEGF189b, which are anti-angiogenic proteins → regulate VEGFxxxa pro-angiogenic activity, make up a higher % of total VEGF-A in quiescent vessels.
Some compounds described as anti-angiogenic are actually partial agonists that find it difficult to stand up when compared to the full agonist VEGF-165-8a.
VEGF-Ax is the newest identified isoform and it contains both 8a and 8b exons. Its physiological function is largely unknown.
Regulation involves RNA binding proteins such as the serine/arginine (SR) rich splicing factors SRSF1, SRSF2, SRSF5 and SRSF6. These are activated via phosphorylation by SR protein kinase 1 (SRPK1). The phosphorylated SRSF translocates to the nucleus and binds regulatory sites at the exon boundaries that triggers exon removal.
Splicing can be influenced by cellular factors such as other growth factors, specific SRSF and cell type specificity of these.
VEGF Receptors
Made up of 7 extracellular IgG-like domains, a TM domain, a juxtamembrane domain, an intracellular split kinase domain, and N and C lobes.
The split intracellular catalytic kinase domain encompasses and ATP binding domain (TKD1), a kinase insert domain (KID) and a phosphotransferase domain (TKD2).
VEGFR1 (Flt-1)
Expressed on vascular endothelial cells and monocyte/macrophage lineage cells where it promotes mobilisation from the bone marrow.
Binds VEGF-A, VEGF-B and PlGF.
Involved in embryonic vascularisation and potentially chemotherapy-induced neuropathic pain - supposed to be downregulated in adults but it becomes upregulated in pathophysiology.
It binds VEGF165a with a 10x higher affinity than VEGFR2.
Weak kinase activity compared to VEGFR2 – ‘antagonises VEGFA/VEGFR2’
Its extracellular domain can be cleaved to produce a soluble VEGF trap, negatively modulating VEGFR2 by decreasing local VEGF-A concentrations - binds VEGF-A but does not produce a signal.
PIGF and VEGF-A signalling through VEGFR1 are linked to neo-vessel formation in some cancers.
VEGFR2
Prototypical VEGF receptor.
Binds VEGF-A and VEGF-D (+ non-human E and F).
Expressed on vascular and lymphatic endothelial cells, with particularly high expression on the leading edge of tip cells in angiogenic sprouting.
Alteration in VEGFR2 expression can result in embryonic lethality or severe cardiovascular abnormalities.
Binds all known VEGF-A isoforms with nanomolar affinities.
Interacts with Neuropilin-1 receptors.
VEGFR3
VEGFR3 is preferentially expressed on lymphatic endothelial cells but expression on vascular endothelial cells can be induced in the developing retina and in cancer angiogenesis.
Binds VEGF-C and VEGF-D.
VEGFR3 is detected in many tumour types and can correlate with lymphatic node metastasis, e.g. prostate, ovarian, lung adenocarcinoma
VEGF-C signals through AKT (PKB) and ERK to promote lymphangiogenesis.
VEGF-D is dispensable during normal lymphatic vessel growth, but its expression significantly contributes to lymphatic metastasis of tumours.
Embryonic lethalithy is observed with deletion of:
- VEGF-C → defects in lymphatic endothelial cell (LEC) migration and lymphatic vessel sprouts
- VEGFR3 → defects in cardiovascular development
VEGF binding and activation
The VEGF dimer binds two VEGFR monomers at the Ig D2 and D3 domains, bringing them closer and allowing dimerisation.
VEGFR2 is activated by VEGF through trans-autophosphorylation.
ATP and Mg2+ are necessary for the intrinsic tyrosine kinase activity of VEGFRs
ATP binds in a cleft between the 2 TKDs, at the glycine rich loop, exposing a stabilising lysine (K868).
Mg2+ is chelated by a DFG motif in the activation loop
Initial auto-phosphorylation occurs at tyrosine residues Y1054/Y1059.
This orientates the activation loop to a conformation that promotes additional auto-transphosphorylation, leading to recruitment of adaptor proteins.
Grb2 is a signalling adaptor protein that contains an SH2 domain that interacts with the phosphotyrosine residues, and two SH3 domains that interact with proline-rich domains found on other adaptors like the SOS, Src or PI3K.
The receptor is able to internalise into intracellular compartments, alongside the ligand → majority of signalling occurs in intracellular compartments, not at the membrane. Ligands may have to be able to enter these compartments to affect the signalling of these receptors.
- Clathrin and AP2 facilitate internalisation
The receptors can be degraded in lysosomes or recycled by reinsertion into the membrane once the ligand unbinds.
VEGFR heterodimers
VEGFR1/2 heterodimers are thought to negatively regulate VEGFR2 homodimers. This may be because R1 has higher affinity for VEGF-A isoforms but lower kinase activity.
VEGFR2/3 heterodimers are involved in angiogenic sprouting and mechanosensitivity.
Neuropilins
Neuropilin 1 and 2 are VEGFR co-receptors.
NRP1 is expressed on arterial but not lymphatic ECs, as well as some immune (macrophages, T-cells) and neuronal (pericytes) cells.
Upregulated in prostate cancer, correlates with aggressiveness and poor patient outcomes.
Major role in axonal guidance due to its role in binding semaphorins (SEMA3a).
NRP2 is expressed on lymphatic and venous ECs. High expression correlates with increased tumour lymphangiogenesis.
Neuropilin receptors bind some VEGF-A isoforms (longer isoforms) with comparable affinity to VEGFR2.
Short cytoplasmic tail allows interaction with PDZ domain containing adaptor proteins
- no kinase activity
- motif required for VEGFR modulation
Modulation of VEGF signalling comes through facilitation of scaffold formation.
Can also act as a co-receptors for other receptors, such as FGFR, PDGFR, MET and complement split proteins.
NRP1 can bind:
- VEGF165a, VEGF189a and VEGF206a, that follow the CendR rule, a position-dependent protein motif that regulates cellular uptake and vascular permeability through interaction with neuropilin-1.
- Cannot bind VEGFb → terminal arginine (R) in VEGFa seems to be important
NRP2 can bind VEGF-A (lower affinity), VEGF-C and VEGF-D.
VEGF binds as a dimer across two neuropilins or by bridging the gap between neuropilin and VEGFRs.
NRP1 also binds PDGFR-β receptors in the presence of the PDGFR-DD ligand dimer.
Neuropilins may bind matrix species, such as proteoglycans. The receptor complex can also interact with synectin and myosin IV.
Neuropilins may alter VEGF trafficking, altering signalling.
- If the receptors are on the same cell membrane, neuropilin can facilitate VEGFR2 internalisation, promoting angiogenic signalling.
- if the receptors are on two different cells, it prevents internalisation and therefore reduces signalling. VEGFR2 is arrested at the cell surface → reduced tumour branching and proliferation.
VEGF signalling in cancer
Tumour growth is limited by a lack of nutrients and oxygen.
Hypoxia and low pH stimulate VEGF secretion from tumour cells, which initiates angiogenesis.
Secreted matrix metalloproteinases (MMPs) degrade the EC basement membrane. This allows ECs to escape the vessel and move towards the tumour resulting in tumour vascularization.
Pericyte recruitment and Notch signalling also occurs.
Tumour vasculature is disorganised and intrinsically leaky, facilitating tumour escape.
- MMP dysregulation causes degradation of the basement membrane
- poor pericyte coverage
- high permeability
- low immune cell extravasation due to the immunosuppressive tumour microenvironment
VEGFR1 and 2 are also expressed on tumour-associated M2 macrophages and are involved in recruitment to the tumour site. These are anti-inflammatory, preventing the immune system from attacking the tumour.
VEGFR3 and VEGF-C are secreted by tumour cells, increasing lymphagiogenesis.
Increased expression of NRP1 correlates with aggressive tumours and upregulation has been observed on cancer stem cells found at the centre of the tumour, suggesting contribution to self renewal and chemoresistance (decreased drug penetration).
Therapeutic targeting of VEGF-A/VEGFR
Monoclonal antibodies are selective. They are efficacious for ~7 months, but resistance can then develop. They also have low stability, are expensive and can cause side effects like hypertension and proteinuria.
Bevacizumab is a humanised VEGF-A monoclonal antibody used for metastatic colorectal cancer, NSCLC and metastatic renal cell carcinoma.
Ramucirumab is a human IgG1 monocloanal antibody against the extracellular domain of VEGFR2, preventing ligand binding and receptor activation. It is used for metastatic NSCLC, colorectal cancer and gastric cancer. However, it increases circulating VEGF levels, which may be an issue.
Small molecular inhibitors are cheaper and there is evidence for their efficacy, but they can have selectivity issues. This leads to systemic side effects, most commonly hypertension, which can be so severe that it causes gut perforation. Resistance can also develop.
Sorafenib is a multi-targeted RTK inhibitor that binds VEGFRs, but also C-Raf, B-Raf and PDGFRs. It is used for renal cell and hepatocellular carcinoma and thyroid cancer. It has antiproliferative and antiangiogenic effects.
Axitinib is a second generation small molecule inhibitor, with more selectivity for VEGFR2 and improved potency. It is a second-line treatment for RCC refractory to multi-target RTKIs.
