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Conformational Nanobodies Reveal Tethered Epidermal Growth Factor Receptor Involved in EGFR/ErbB2 Predimers
- Source :
- ACS Nano, ACS Nano, American Chemical Society, 2015, 9 (2), pp.1388-99. ⟨10.1021/nn505752u⟩, ACS Nano, 2015, 9 (2), pp.1388-99. ⟨10.1021/nn505752u⟩
- Publication Year :
- 2015
- Publisher :
- American Chemical Society (ACS), 2015.
-
Abstract
- International audience; The epidermal growth factor receptor (EGFR) is a cell-surface receptor with a single transmembrane domain and tyrosine kinase activity carried by the intracellular domain. This receptor is one of the four members of the ErbB family including ErbB2, ErbB3, and ErbB4. Ligand binding, like EGF binding, induces a conformational rearrangement of the receptor and induces a homo/hetero dimerization essentially with ErbB family receptors that leads to the phosphorylation of the kinase domain, triggering a signaling cascade. EGFR can also form inactive dimers in a ligand-independent way through interactions between cytoplasmic domains. To date, the conformation of EGFR extracellular domain engaged in these inactive dimers remains unclear. In this study, we describe the successful selection and characterization of llama anti-EGFR nanobodies and their use as innovative conformational sensors. We isolated three different specific anti-EGFR clones binding to three distinct epitopes. Interestingly, the binding of all three nanobodies was found highly sensitive to ligand stimulation. Two nanobodies, D10 and E10, can only bind the ligand-free EGFR conformation characterized by an intramolecular tether between domains II and IV, whereas nanobody G10 binds both ligand-free and ligand activated EGFR, with an 8-fold higher affinity for the extended conformation in the presence of ligand. Here we took advantage of these conformational probes to reveal the existence of tethered EGFR in EGFR/ErbB2 predimers. These biosensors represent important tools allowing the determination of EGFR conformations and should help the design of relevant inhibitors.
- Subjects :
- [SDV.BIO]Life Sciences [q-bio]/Biotechnology
MESH: Protein Structure, Quaternary
Receptor, ErbB-2
General Physics and Astronomy
Biosensing Techniques
Epitopes
Mice
Antibody Specificity
MESH: Animals
General Materials Science
ERBB3
Epidermal growth factor receptor
conformational changes
ERBB4
biology
MESH: Protein Multimerization
General Engineering
Ligand (biochemistry)
epidermal growth factor receptor (EGFR)
nanobodies
ErbB Receptors
MESH: Receptor, ErbB-2
Transmembrane domain
Biochemistry
MESH: Camelids, New World
phage display
Camelids, New World
Tyrosine kinase
MESH: Biosensing Techniques
Binding domain
MESH: Epitopes
homogenous time-resolved fluorescence
MESH: Receptor, Epidermal Growth Factor
MESH: Single-Domain Antibodies
Cell Line
single domain antibodies
Animals
Humans
MESH: Antibody Specificity
Protein Structure, Quaternary
MESH: Mice
Binding Sites
MESH: Humans
Single-Domain Antibodies
biosensors
MESH: Cell Line
MESH: Binding Sites
Protein kinase domain
biology.protein
Biophysics
Protein Multimerization
Subjects
Details
- ISSN :
- 1936086X and 19360851
- Volume :
- 9
- Database :
- OpenAIRE
- Journal :
- ACS Nano
- Accession number :
- edsair.doi.dedup.....64a77e3270845ee654e0a19ba2101ba1