Receptor Tyrosine Kinases

Question 1

RTKs general

Answer 1

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.

Question 2

RTK families

Answer 2

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.

Question 3

RTK dimerisation

Answer 3

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.

Question 4

PDGFRs

Answer 4

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.

Question 5

VEGFR subtypes

Answer 5

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.

Question 6

Cross RTK family interactions and co-receptors

Answer 6

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).

Question 7

Effect of growth factors on extracellular receptor regions

Answer 7

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.

Question 8

Eph/EPHRIN signalling

Answer 8

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.

Question 9

Activation of intracellular kinase domains

Answer 9

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.

Question 10

EGFR signalling pathways and adaptor proteins

Answer 10

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.

Question 11

Alpha screen

Answer 11

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.

Question 12

Reporter genes

Answer 12

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.

Question 13

VEGF basics

Answer 13

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.

Question 14

VEGF-A structure

Answer 14

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.

Question 15

VEGF-A isoforms

Answer 15

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.

Question 16

VEGF Receptors

Answer 16

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).

Question 17

VEGFR1 (Flt-1)

Answer 17

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.

Question 18

VEGFR2

Answer 18

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.

Question 19

VEGFR3

Answer 19

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

Question 20

VEGF binding and activation

Answer 20

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.

Question 21

VEGFR heterodimers

Answer 21

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.

Question 22

Neuropilins

Answer 22

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.

Question 23

VEGF signalling in cancer

Answer 23

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).

Question 24

Therapeutic targeting of VEGF-A/VEGFR

Answer 24

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.

Question 25

Therapeutic targeting of NRP1

Answer 25

We could target Neuropilin receptors, but therapeutics seem to have toxicity and affinity issues.
NRP1 can bind CRN peptides that facilitate endocytosis - these could be used for delivery of cytotoxic drugs.
Dual/combination therapies could be used, e.g. alongside inhibitors of EGFR, PD-1, Integrins and PIM-1 kinases.

Question 26

EGFRs

Answer 26

The human epidermal growth factor receptor family consists of four closely related RTKs: EGFR (ErbB1; HER1), HER2 (ErbB2), HER3 (ErbB3), HER4 (ErbB4).
EGFR, HER2, HER3 are all implicated in the development and progression of cancer. The role of HER4 is less clear.
Agonist binding elicits dimer formation as the dimerisation domain unfolds, leading to intracellular signalling, particularly through MAPK and PI3K-Akt pathways.
BRET can be used to monitor conformation changes - nanoluciferase is attached to the far end of the receptor and a fluorescent nanobody is added to a medial part. Fluorescence is emitted as the nanobody binds to the nanoluciferase when EGF binds and a conformational change occurs.
HER2 is distinct from the other receptor as it has no identified ligand. It is naturally in an open configuration, ready to dimerise with other monomers → constitutively active. Overexpression is implicated in cancer.
HER3 does not have an active kinase domain → cannot initiate signalling on its own, needs to form heterodimers with other members of the family.

Question 27

EGFR ligands and signalling

Answer 27

There are 7 known EGFR ligands:
- Epidermal growth factor (EGF)
- Transforming growth factor-alpha (TGF-α)
- Heparin-binding EGF-like growth factor (HB-EGF)
- Amphiregulin (AREG)
- Epiregulin (EREG)
- Epigen (EPGN)
- Betacellulin (BTC)
Neuregulins can also interact with EGFRs.
All are synthesised as type 1 TM precursors that have to be enzymatically cleaved to initiate signalling.
Autocrine signalling occurs when a ligand is released from a cell and binds to EGFRs on the same cell.
Paracrine signalling refers to the released ligand acting on a nearby cell.
Juxtacrine signalling is when a non-cleaved transmembrane ligand binds to an EGFR on an adjacent cell when they come into contact (e.g. HB-EGF).
AREG, EGF, TGFa and HB-EGF can be packaged into exosomes that can be taken up by neighbouring cells, leading to exosomal targeted receptor activation. This can be seen in cancer:
- Tumour releases exosomes containing receptors and membrane-bound growth factors following activation.
- Vesicles move around the body
- Recipient cells take these up by membrane fusion or endocytosis, which can lead to further tumour generation.

Question 28

Heparin-binding EGF-like growth factor (HB-EGF)

Answer 28

HB-EGF is released from membrane-bound pro-ligands by the action of matrix metalloproteinases (MMPs) or A disintegrin and metalloproteinases (ADAMs).
Transactivation on one signalling pathway involving these enzymes leads to activation of EGFR signalling.
MMPs/ADAMs are activated via GPCR signalling.
Soluble mature HB–EGF is a potent mitogen and chemotactic factor for fibroblasts and smooth muscle cells.
HB–EGF activates HER1 and HER4 and binds to cell-surface heparan sulphate proteoglycans (HSPGs).

Question 29

Dimerisation

Answer 29

EGF is monomeric. Binding induces a conformational change in the receptor that exposes a dimerisation domain.
HER2 has no known ligand and the dimerization domain is exposed in the resting state. It readily dimerises with other receptors but can also form homodimers and be active without binding a ligand.
HER3 has no active kinase domain and must form heterodimers to initiate signalling.
EGFR homodimers and EGFR/HER2 can initiate MAPK signalling.
HER2-containing dimers can initiate PI3K signalling.

Question 30

EGFR and cancer - mutations

Answer 30

Activating mutations arise within the catalytic tyrosine kinase domain.
L858R and L861Q mutations occur within Exon 21, which encodes the activation loop.
Short in-frame deletions within Exon 19, e.g. E746-A750del within the kinase domain, leads to a loss of five amino acids and increased tyrosine kinase activity.

Question 31

EGFR, cancer and treatment

Answer 31

EGFR is overexpressed in 60% of cases of non-small cell lung carcinoma (NSCLC), which accounts for 85% of all lung cancers.
Erlotinib and gefitinib are reversible, type 1 inhibitors, binding to the ATP binding site. Afatinib is an irreversible inhibitor at this site. Lapatinib binds to a site adjacent to the kinase domain (type 2 inhibitor) and keeps it in an inactive conformation through allosteric modulation.
These types of inhibitors are easy and cheap to develop and initially effective, but as the ATP binding site is highly conserved, they lack selectivity, which can cause side effects. They also have to compete with high intracellular concentrations of ATP. Resistance also quickly develops.
Third-generation covalent inhibitors like Rocilatinib are able to bind to receptors that have acquired the T790M gatekeeper mutation involved in resistance.
These are all small molecular inhibitors.

Question 32

EGFR TKD activation

Answer 32

The EGFR TKD is allosterically activated by contacts between the C-lobe of the activator TKD and N-lobe of the receiver TKD.
The activator TKD destabilises auto-inhibitory interactions that involve the activation loop of the receiver.
In this asymmetrical dimer, the C-terminal lobe of one kinase pushes the αC helix on the N-terminal lobe of the other kinase towards the active site.
ErbB3/HER3, which lacks active kinase activity, can still be involved in activating the kinase of other Erb receptor members via dimerization.

Question 33

HER2 and cancer

Answer 33

15% of breast cancers have gene amplification or overexpression of HER2, resulting in a more aggressive phenotype and poor prognosis. Aberrant HER2 expression is also found in gastric, ovarian and salivary gland tumours.
HER2 overexpression is associated with aggressive tumour behaviour, including a higher grade, increased mitotic count, positive lymph node metastases, and a worse survival outcome.
Lapatinib is a type 2 receptor tyrosine kinase inhibitor against EGFR and HER2.
Trastuzumab (Herceptin) is a recombinant humanised monoclonal antibody directed against the cysteine-rich extracellular domain IV of HER2. It may cause cardiotoxicity.
Pertuzumab is another recombinant humanised antibody, but it is directed against the extracellular dimerisation domain (domain II) of HER2, preventing dimerisation with other monomers, such as HER3.
Antibody therapeutics cause the immune system to attack cells expressing HER2, causing immune-mediated destruction of cancer cells.
Antibody therapeutics are expensive, unstable, can have penetrance and delivery issues and may lead to side effects like hypertension and cardiotoxicity.
Ado-trastuzumab emtansine (T-DM1) is an antibody-drug conjugate that combines trastuzumab with the potent cytotoxic agent, emtansine. T-DM1 delivers targeted chemotherapy directly to HER2-expressing cancer cells, reducing systemic toxicity.
These treatments are only effective in HER2+ breast cancer → overexpression has to be present. This can be tested using immunohistochemistry, flourescence in-situ hybridization, ELISA and mRNA measurements.
Trifunctional antibodies like ertumaxomab target HER2 and engage T-cells to enhance antitumour immunity.

Question 34

HER3 and Signalling Pathways in Cancer

Answer 34

There are two key signalling pathways:
- MAPK (ERK), which stimulates tumour cell proliferation
- PI3K-Akt which promotes tumour cell survival
Receptor activation facilitates recruitment of a signalling complex.
Although HER3 (ErbB3) lacks innate kinase activity, it can heterodimerize with other ErbB receptors.
ErbB3 is frequently expressed in human mammary tumours. Overexpression of the ErbB3 agonist heregulin results in increased tumour formation.

HER3 seems to be the preferred dimerization partner when signalling occurs through the PI3K pathway, which is important for tumour cell survival. The HER2/HER3 heterodimer is considered the most potent Erb pair.

Question 35

EGFR extracellular domain stabilisation and biased signalling

Answer 35

Structural studies indicate that EGFR ligands differentially stabilise the extracellular domains in receptor dimers.
A single ligand-bound dimer shows negative cooperativity across the dimer interface, resulting in reduced affinity for EGF binding to the second promoter. This is an asymmetric dimer interface.
Symmetric dimers occur when two ligands are bound. These are strong.
Asymmetric dimers are weak. They can also occur when a partial agonist binds. These show different signalling characteristics (biased signalling).
Different EGFR ligands stabilise dimers with distinct structures.
Epiregulin (EREG) and epigen (EPGN) induce weaker EGFR dimers than EGF and TGF-α. Unexpectedly, the weaker dimerization elicits more sustained EGFR signalling. This provokes responses in breast cancer cells associated with differentiation rather than proliferation, indicating a possible ligand bias.
Glioblastoma mutations (e.g R84K) in the extracellular domains prevent EGFR from discriminating between its activating ligands. These mutations allow EREG and other low affinity ligands to form strong EGF-like dimers → increased proliferation as cells do not differentiate.

The stability of the ligand-receptor complex influences the duration of active signalling, a concept known as kinetic proofreading. Unstable complexes dissociate or are dephosphorylated rapidly, limiting downstream signalling, while stable complexes allow for prolonged activation and recruitment of downstream effectors.
Different ligands can also induce disting juxtamembrane domain conformations.

Question 36

EGFRs and membrane lipids *

Answer 36

The membrane environment is important for EGF regulation. Membrane lipids can interact with both the intracellular and extracellular regions of EGFR, influencing its activity.

Question 37

Transactivation of GPCRs and RTKs *

Answer 37

GPCRs and RTKs interact and influence each other’s signalling pathways.
GPCR activation can lead to tyrosine phosphorylation of RTKs.
Ligand-dependent transactivation involves MMPs or ADAMs, which are activated by GPCR signalling through Gβγ subunits or Src. These then cleave EGFR pro-ligands. The cleaved ligands then bind to RTKs on the cell surface, triggering downstream signalling.
In ligand-independent transactivation GPCRs activate RTKs indirectly through intracellular protein kinases like Src, PI3K, and PYK. For example, Src phosphorylates EGFR following activation of the corticotropin-releasing factor receptor 1.
Transactivation can be bidirectional, with RTKs influencing GPCR signalling through:
- Tyrosine phosphorylation of GPCRs and GRKs.
- Modulation of GPCR serine/threonine phosphorylation.
- Altered effector protein coupling to the GPCR