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Bipartite tetracysteine display reveals allosteric control of ligand-specific EGFR activation.

ABSTRACT: Aberrant activation of the epidermal growth factor receptor (EGFR), a prototypic receptor tyrosine kinase, is critical to the biology of many common cancers. The molecular events that define how EGFR transmits an extracellular ligand binding event through the membrane are not understood. Here we use a chemical tool, bipartite tetracysteine display, to report on ligand-specific conformational changes that link ligand binding and kinase activation for full-length EGFR on the mammalian cell surface. We discover that EGF binding is communicated to the cytosol through formation of an antiparallel coiled coil within the intracellular juxtamembrane (JM) domain. This conformational transition is functionally coupled to receptor activation by EGF. In contrast, TGF? binding is communicated to the cytosol through formation of a discrete, alternative helical interface. These findings suggest that the JM region can differentially decode extracellular signals and transmit them to the cell interior. Our results provide new insight into how EGFR communicates ligand-specific information across the membrane.

SUBMITTER: Scheck RA

PROVIDER: S-EPMC3789377 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Bipartite tetracysteine display reveals allosteric control of ligand-specific EGFR activation.

Scheck Rebecca A RA Lowder Melissa A MA Appelbaum Jacob S JS Schepartz Alanna A

ACS chemical biology 20120605 8

Aberrant activation of the epidermal growth factor receptor (EGFR), a prototypic receptor tyrosine kinase, is critical to the biology of many common cancers. The molecular events that define how EGFR transmits an extracellular ligand binding event through the membrane are not understood. Here we use a chemical tool, bipartite tetracysteine display, to report on ligand-specific conformational changes that link ligand binding and kinase activation for full-length EGFR on the mammalian cell surface ...[more]

PMID: 22667988

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Project description:BackgroundMagnetic nanoparticles (NPs) are of particular interest in biomedical research, and have been exploited for molecular separation, gene/drug delivery, magnetic resonance imaging, and hyperthermic cancer therapy. In the case of cultured cells, magnetic manipulation of NPs provides the means for studying processes induced by mechanotransduction or by local clustering of targeted macromolecules, e.g. cell surface receptors. The latter are normally activated by binding of their natural ligands mediating key signaling pathways such as those associated with the epidermal growth factor (EGFR). However, it has been reported that EGFR may be dimerized and activated even in the absence of ligands. The present study assessed whether receptor clustering induced by physical means alone suffices for activating EGFR in quiescent cells.Methodology/principal findingsThe EGFR on A431 cells was specifically targeted by superparamagnetic iron oxide NPs (SPIONs) carrying either a ligand-blocking monoclonal anti-EGFR antibody or a streptavidin molecule for targeting a chimeric EGFR incorporating a biotinylated amino-terminal acyl carrier peptide moiety. Application of a magnetic field led to SPION magnetization and clustering, resulting in activation of the EGFR, a process manifested by auto and transphosphorylation and downstream signaling. The magnetically-induced early signaling events were similar to those inherent to the ligand dependent EGFR pathways. Magnetization studies indicated that the NPs exerted magnetic dipolar forces in the sub-piconewton range with clustering dependent on Brownian motion of the receptor-SPION complex and magnetic field strength.Conclusions/significanceWe demonstrate that EGFR on the cell surface that have their ligand binding-pocket blocked by an antibody are still capable of transphosphorylation and initiation of signaling cascades if they are clustered by SPIONs either attached locally or targeted to another site of the receptor ectodomain. The results suggest that activation of growth factor receptors may be triggered by ligand-independent molecular crowding resulting from overexpression and/or sequestration in membrane microdomains.

Dataset Information

Bipartite tetracysteine display reveals allosteric control of ligand-specific EGFR activation.

Publications

Bipartite tetracysteine display reveals allosteric control of ligand-specific EGFR activation.

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