Pharm 4620 Video Assignment
Topic: Structural basis of the activation of c-MET receptor
Paper: https://pubmed.ncbi.nlm.nih.gov/34210960/
1. Contains a descriptive title.
Investigating the signalling pathway of the c-MET receptor and the structural basis of its
activation
2. Introduces the molecules involved in the process.
The c-Met Receptor is a receptor tyrosine kinase (RTKs). RTKs are enzymes which catalyze the
phosphorylation of tyrosine residues using ATP. RTKs regulate many essential cellular processes
in mammalian development, cell function and tissue homeostasis by mediating cell proliferation,
differentiation, migration, metabolism, and angiogenesis.
The c-MET receptor is composed of a Semaphorin (SEMA) domain, a plexin-semaphorinintegrin (PSI) domain, four consecutive immunoglobulin-plexin-transcription factors (IPT1-4)
domains in the extracellular region, a single transmembrane helix (TM), the juxtamembrane,
followed by an intracellular tyrosine kinase catalytic domain, and the docking site with carboxyterminal sequences.
c-MET is initially expressed as a single-chain precursor, which then turns into the mature form,
which contains α and β subunits, through proteolytic cleavage between Arg307 and Ser308.
c-MET can be activated by either - hepatocyte growth factor - HGF or the natural isoform of
HGF - NK1.
HGF is produced by stromal cells. When bound to c-MET, HGF stimulates epithelial cell
proliferation, morphogenesis and motility. Similar to its receptor c-MET, HGF is secreted as a
single chain or biologically inert precursor and is converted into its bioactive form through
proteolytic cleavage between Arg494 and Val495. The mature form of HGF consists of an α- and
β-chain, which are held together by a disulphide bond. The α-chain contains an N-terminal
hairpin loop followed by four kringle domains. The β-chain contains the SPH domain. When
bound to c-MET, HGF induces the dimerization of c-MET which enables the intracellular kinase
domains (KDs) to undergo autophosphorylation. The phosphorylated kinase domains recruit
cytosolic effector proteins, which causes the activation of downstream signaling pathways. Both
the pre-mature and cleaved HGF can bind to c-MET with high affinity, but only the cleaved HGF
can activate the signalling pathway – which is why proteolytic cleavage is a critical step.
c-MET could also be activated by NK1, which is a naturally occurring isoform of HGF (through
alternative splicing). NK1 itself forms a stable dimer that can recruit two c-MET for activation.
The two c-METs are placed in relatively close proximity and are bound symmetrically, which
enables their intracellular kinase domains to undergo autophosphorylation and initialize
downstream signaling.
Heparin, which is a glycosaminoglycan can promote HGF-induced c-MET activation by
enhancing the binding affinity between HGF and c-MET, and NK1 and c-MET.
3. Shows/mentions the tissues and cell types where your signaling pathway functions.
c-MET is usually expressed in stem cells, progenitor cells and the epithelial cells of several
organs, such as the liver, pancreas, kidney, muscle and bone marrow. c-MET receptor activation
stimulates organogenesis, wound healing, embryonic development and cell migration. During
embryogenesis, c-MET and HGF provide essential signals for survival and proliferation of
hepatocytes and placental trophoblast cells.
4. Indicates where in the cell your signaling molecules are localized.
c-MET is a cell surface receptor which can be found spanning the plasma membrane. Its ligands –
HGF or NK1 bind to it extracellularly. HGF is secreted into the extracellular matrix by stromal
cells and undergoes proteolytic cleavage by an extracellular protease before becoming binding to
c-MET. Heparin, the biomolecule which promotes the binding of HGF and NK1 to c-MET, also
binds to the extracellular domains of c-MET. The kinase domains in c-MET which undergo
autophosphorylation are located on the cytoplasmic side of the membrane.
5. Conveys the cellular effect(s) of your signaling pathway.
This flowchart maps out all the signal pathways induced by c-MET activation.
c-MET activation causes the binding of SHC and GRB2, which recruits guanine nucleotide
exchanger SOS which in turn stimulates the activity of the rat sarcoma viral oncogene homolog
(RAS). This leads to the indirect activation of RAF kinases, which subsequently activate the
MAPK effector kinase MEK and finally MAPK, which then translocate to the nucleus to activate
transcription factors responsible for regulating genes involved in cell proliferation, cell motility
and cell cycle progression. These genes are also regulated by the binding of SHP2 to c-MET
The binding of P13K to c-MET, induces a signal through AKT/protein kinase B, which stimulates
the signal cascade responsible for cell survival. Cellular migration is also mediated downstream
of c-MET by focal adhesion kinase (FAK), which is activated through phosphorylation by SRC
family kinases.
SRC activation can also positively regulate c-MET activation.
Negative regulation of the c-MET receptor is critical to prevent cells from undergoing
uncontrolled proliferation. The Y1003 site is a negative regulatory site for c-MET signaling that
acts by recruiting c-CBL. Regulation of c-MET signaling is also accomplished via its binding to
various protein tyrosine phosphatases (PTPs), which cause dephosphorylation of either the
tyrosines in the kinase domain or the docking tyrosines.
6. Shows the protein‐protein interactions involved in the signaling. Shows any other biomolecular
interactions involved in the signaling.
There are at least two distinct c-MET binding sites on HGF: one is at the α-subunit of HGF, and
the other is located in the SPH domain, which is the only domain existing in the β-subunit of
HGF. The binding of HGF I to c-MET I is mediated by the interaction between the N, K2, K3,
K4 and SPH domains of HGF I, which are arranged as a C-clamp shape that pack tightly against
the SEMA domain of c-MET 1. In total, there are four interfaces are formed between c-MET I
and HGF I. HGF I bound to c-MET1 enables the dimerization of the two c-MET molecules which
is necessary for receptor activation. The binding of HGF I to c-MET II is mediated by the K1
domain located on the opposite side of HGF I, which makes direct contact with the SEMA
domain of c-MET II. This minimal 2:1 ratio of c-MET:HGF active complex is further stabilized
by a second HGF molecule and heparin, leading to a more stable 2:2 complex and enhanced
activation of c-MET. HGF II binding strengthens the interaction between HGF I and c-MET II
and thereby stabilizes the entire c-MET I/HGF I/c-MET II complex. Although the surface of the
newly bound second HGF II K1 domain is free for c-MET binding, the binding of another c-MET
to HGF II-K1 in such rigid conformation would clash with HGF I bound c-MET II, which is why
the 2:2 complex cannot recruit additional c-MET molecule.
The structural model of the 2:2 c-MET:NK1 (the natural isoform of HGF) complex is distinct
from the way that HGF activates c-MET, as NK1 forms a stable head-to-tail dimer and recruits
two c-METs in a symmetric manner. The two c-MET molecules bound with the NK1 dimer are
placed in close proximity, which enables their intracellular KDs to undergo autophosphorylation
and initialize downstream signaling.
A biomolecule that is crucial for this signalling pathway is the glycosaminoglycan – heparin.
Heparin binds concurrently to the extended loop or the IPTI domain of c-MET II and the N
domain HGFI/NK1, and acts as a “glue” between both domains. The binding of heparin enhances
the binding affinity between HGF/NK1 with C-MET, stabilizes the receptor-ligand complex and
promotes the dimerization of c-METs.
7. Shows how the cellular localization of the biomolecules may change during the signaling process.
Throughout the signalling process, the ligands are localized to the extracellular domains of the cMET receptors; whereas the cytosolic effector proteins which are recruited by the phosphorylated
tyrosine residues remain bound to the intracellular domains of c-MET. Following negative cMET regulation, the ligands and cytosolic proteins disassociate from the receptor, while c-MET
remains localized to the cell membrane.
8. Indicates how the pathway is dysregulated or dysfunctional associated with a disease.
The c-MET pathway results in cell proliferation, morphogenesis and motility - all processes
which could lead to the development of tumors and cancer progression, when upregulated. The
enhanced activation of c-MET may be induced by the occurrence of gene amplifications,
activating/inhibiting mutations; or via transcriptional upregulation of the c-MET protein.
Gene Amplification: In hereditary cancers, heterozygous mutations are usually accompanied by
trisomy of whole chromosome 7. The c-MET gene is located on chromosome 7q21-31, trisomy of
this chromosome would cause amplification of the c-MET gene.
Mutations: Evidence of mutations in the c-CBL binding site of the juxtamembrane domain
(which acts as a negative regulatory site for c-MET signaling) and in the HGF-binding region of
the SEMA domain (a primary step in the activation of the c-MET signal cascade) have been
found in multiple human cancers.
Transcriptional Upregulation: Increased protein expression as a consequence of transcriptional
upregulation is the most frequent cause of constitutive c-MET activation in human tumors.
Growing tumors tend to cause hypoxia due to the lack of oxygen diffusion to the centre of a
tumor. Hypoxia has been found to be a mechanism by which c-MET transcription is activated. It
upregulates the gene through the binding of intracellular oxygen dependent - transcription factor
HIF1 α.
9. Includes at least ONE three‐dimensional molecular structure image of a component of this
signaling pathway.
This is a 3-Dimensional Molecular Structure of …
10. Describes how you would
a. design a new drug to disrupt a particular aspect of the signaling based on the protein
structure to treat disease.
Current drugs used to treat tumor development and cancer progression, target c-MET by
stabilizing its inactive form or by binding to its natural ligand – HGF and prevent its
binding to the receptor. One potential mode of therapy which could be used to treat the
prolonged activation of the c-MET receptor, could be the stimulation of the negative cMET regulation pathway. The negative feedback loop of c-MET is evidently suppressed
in tumor cells. Therefore, a new drug could be targeted at the tissue of interest, to induce
the transcriptional upregulation of c-CBL (ubiquitin ligase casitas B-lineage lymphoma).
The recruitment of c-CBL to the Y1003 negative regulatory site in the juxtamembrane of
receptor, initiates the negative feedback signalling of c-MET. Transcription upregulation
of the c-CBL gene would increase the amount of ligand available to bind to the negatives
regulatory site, which would in turn downregulate c-MET activity and decease the
progression of the tumor/cancer, until the site becomes fully saturated.
11. Includes credits showing references and information sources used
Goździk-Spychalska, J., Szyszka-Barth, K., Spychalski, L., Ramlau, K., Wójtowicz, J., BaturaGabryel, H., & Ramlau, R. (2014). C-MET inhibitors in the treatment of lung cancer. Current
treatment options in oncology, 15(4), 670–682. https://doi.org/10.1007/s11864-014-0313-5
Organ, S. L., & Tsao, M. S. (2011). An overview of the c-MET signaling pathway. Therapeutic
advances in medical oncology, 3(1 Suppl), S7–S19. https://doi.org/10.1177/1758834011422556
Uchikawa, E., Chen, Z., Xiao, G. Y., Zhang, X., & Bai, X. C. (2021). Structural basis of the
activation of c-MET receptor. Nature communications, 12(1), 4074. https://doi.org/10.1038/s41467021-24367-3
Western Blot