Molecular Cell Biology

Prof. D. Karunagaran
Department of Biotechnology
Indian Institute of Technology Madras

Module 7
Cell Signaling Mechanisms
Lecture 7
RTK Signaling








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RTK- Receptor Tyrosine Kinase
Receptor Tyrosine Kinases (RTKs) are one of the most well-studied kinases
known to be involved in signaling
RTKs control cell cycle, proliferation, differentiation, survival and metabolism of
cells
50 RTKs have been identified in humans so far
RTKs generally contain an extracellular ligand binding domain, single
transmembrane helix and a cytoplasmic domain containing one or more tyrosine
residues at their carboxy terminal tails
Tyrosine kinase activity resides in the cytoplasmic domain
Mutations in RTKs are found in cancer, diabetes, atherosclerosis, inflammation
etc



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Receptor activation
Generally when a growth factor binds to the RTK, the receptor dimerizes
Activation of the receptor requires ligand binding to form an active dimer or
oligomer
Interaction of bivalent ligand to two receptors results in dimerization. example
stem cell factor receptor KIT, Flt1 VEGF receptor and NGF/neurotrophin receptor
TrkA
Ligand mediated dimerization is observed in TrkA
Ligand mediated dimerization with receptor contact is found in KIT
EGFR/ErbB family
Extracellular region contains four domains.
Domains I and III are 160 amino acids in length with helix LRR like solenoid
domains
Both receptors bind to activating ligands
Domain II and IV are rich in cysteine residues
Even though ligand is bivalent it contacts two different sites in a single receptor
rather crosslinking the receptors
When a ligand binds to the receptor, conformational changes occur in the
extracellular domain of EGFR that reveals the dimerization domain II to interact
with other receptor.
Acti vation of Intracellular Kinase Domains
Tyrosine kinase domain (TKD) contains N-lobe and a C-lobe
Activation loop and alphaC helix are the key regulatory elements in N-lobe of
kinase undergoing a configuration change leading to catalysis of
phosphotransfer.
Inactive TKD is cis-autoinhibited by two specific intramolecular interactions
When ligand induces receptor dimerization, this cis-autoinhibition is released
Autoinhibition of TKD by activation loop: Insulin receptor
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Tyrosine in the activation loop of insulin receptor TKD protrude into the active site
to be autophosphorylated by its own kinase domain
This stabilizes configuration of activation loop leading to occlusion of active site
leading to blockage of ATP
When insulin interacts with the receptor tyrosine in the loop is phosphorylated as
a result of dimerization removing the autoinhibition
Autoinhibition of Juxtamembrane region
Phosphorylated activation loop is required to stabilize activated configuration and
for destabilization of cis-autoinhibitory interactions
J uxtamembrane region interacts with various regions of TKD that includes
activation loop and stabilization of autoinhibited conformation
Autoinhibitory interactions are via major tyrosines in juxtamembrane region.
Mutation in this region leads to constitutive activation of RTK majorly found in
cancer
TKD is activated allostericall y
During auto inhibition, TKD has certain level of tyrosine kinase activity for
transphosphorylation of its partner in RTK dimer stabilized by ligand binding
Lead to the competency in phosphorylating its partner and susceptibility to
phosphorylation
When two TKDs in a stabilized dimer are brought together it increases their
respective local concentrations and promotes allosteric effects leading to the
activation of TKD by transiently active TKD
But in EGFR/ErbB TKD forms asymmetric dimer during which C-lobe of one TKD
known as activator interacts with N-lobe of second TKD known as receiver
leading to conformational changes in the N-lobe of the receiver kinase disrupting
cis-autoinhibitory contacts seen in monomer
This results in change in the conformation of receiver kinase to active form
without activation loop phosphorylation
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EGFR also activated allosterically by the receiver kinase in the juxtamembrane
region, which holds the C-lobe activation kinase in the dimer promoting
dimerization and allosteric activation of receiver
Cell signaling and RTK activation
RTKs primary substrates are their respective receptors
Autophosphorylation region in kinase domain has the main regulatory role.
Phopshorylation recruits downstream signaling molecules to receptor.
Trans and autophosphorylation follows specific order
Phases of signaling
Phase I: When ligand binds to receptor autophosphorylation takes place
increasing the catalytic activity of the kinase
Phase II: this recruits signaling components containing Src homology-2 and
phosphotyrosine binding domains
Phase III: Occurs in a few RTKs, Trans-phosphorylation events maximize the
ability of kinase for the phosphorylation of downstream targets
The recruitment of signaling molecules at phase II can also be carried out
through docking proteins
Docking proteins contains membrane-targeting signal at N-terminus followed by
many tyrosine phosphorylation sites serving as a binding sites for many
downstream signaling proteins. Example FRS2 family of proteins
RTK positi ve feed back regulation
Autophosphorylation is reversed by many protein tyrosine phosphatases (PTPs)
Activation of RTK involves ligand initiated kinase activity, ligand inhibited PTP
activity or both
Continuous activation of EGFR by ligand is accompanied by production of
hydrogen peroxide , ROS which is mediated via PI-3K and activated of NADPH-
oxidase
ROS inhibit PTP activity via oxidizing cysteine in active site of phosphatase.
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Another mechanism involves Gab1 which is recruited EGFR through Grb2
adaptor
Activates PI-3K and stimulates Akt-dependent anti-apoptotic signals
EGFR driven activation of Shc and Src promoting ADAM family proteases
cleaving membrane bound heparin-binding EGF-like growth factor from cell
surface. This activates autocrine EGFR signaling in cancer.
RTK negative feedback regulation
Direct activation of PTPs- some PTPs like Shp1 and 2 containing SH2 domain
are recruited to to activated EGFR resulting in dephosphorylation.
Stimulation of EGFR activates protein kinase C that phosphorylates
juxtamembrane domain of EGFR eliminating binding of EGF to EGFR thus
blocking the EGFR activation.
Phosphorylation of docking proteins by MAPK
Down-regulation and signaling via receptor endocytosis
Endocytosis of receptor and ligand occurs followed by ligand stimulation
This results in degradation of both ligand and receptor molecules.
After EGF and EGFR interaction both ligand and receptors are internalized
byclathrin-mediated endocytosis.
In some cases clathrin independent mechanism also occurs.
For trafficking of EGF and EGFR into lysosomes EGFR kinase activity is required
After internalization these activated RTKs are dephosphorylated and
ubiquitylated followed by dissociation of ligand and receptor at very low pH in
endosomal lumen. RTKs may be recycled to membrane.




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Deregulation of RTK in diseases
Deregulation is majorly through mutations
EGFR is truncated and constitutively active in tumors leading to constitutive
activation of EGFR tyrosine kinase
KIT mutations are observed in tumors. TKD domain and intracellular
juxtamembrane segment and extracellular ligand binding regions are mutated in
hematopoietic cancers and gastrointestinal cancers
These mutations stabilize intermolecular interactions between two KIT D5
domains leading to constitutive receptor-mediated dimerization by stabilizing
receptor-receptor contacts to activate the receptors without ligands
Mutations and alterations in EGFR/ErbB famil y
Family of EGFR receptors are mutated and overexpressed in most human
cancers
Majorly ErbB2, an orphan receptor is highly expressed in breast cancer.
In glioblastomas EGFR gene is highly expressed in both wild type and mutated
form.
Mutations can regulate the activation of the promoter by weakening the auto
inhibition








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Targeting RTKs in cancer and other diseases
Two categories
1. Small-molecule inhibitors: targets ATP binding site of the intracellular TKD
Examples: imatinib(Gleevec) inhibits PDGFR, KIT and non-tyrosine kinases Abl
in leukemia.
Sunitinib blocks KIT, VEGFR2, PDGFR, Flt3 tyrosine kinases
Erlotinib and gefitinib inhibits EGFR
2. Monoclonal Antibodi es: Interferes RTK activation and targets RTK- expressing
cells for destruction mediated through immune system
Examples: Herceptin also known as trastuzumab, monoclonal antibodies against
ErbB2
Cetuximab/Erbitux and panitumumab/vectibix monoclonal antibodies against
EGFR
Study Questions
1. What are RTKs?
2. Write a note on different domains of RTKs
3. RTK signaling is initiated by binding of ligand to a) ligand b) receptor
c) cytoplasmic proteins d) none of the above
4. Match the following
imatinib(Gleevec) EGFR
Erlotinib ErbB2
Herceptin leukemia
Cetuximab Small molecule inhibitor
5. .is reversed by PTPs
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