Dataset Information

Multisite EGFR phosphorylation is regulated by adaptor protein abundances and dimer lifetimes.

ABSTRACT: Differential epidermal growth factor receptor (EGFR) phosphorylation is thought to couple receptor activation to distinct signaling pathways. However, the molecular mechanisms responsible for biased signaling are unresolved due to a lack of insight into the phosphorylation patterns of full-length EGFR. We extended a single-molecule pull-down technique previously used to study protein-protein interactions to allow for robust measurement of receptor phosphorylation. We found that EGFR is predominantly phosphorylated at multiple sites, yet phosphorylation at specific tyrosines is variable and only a subset of receptors share phosphorylation at the same site, even with saturating ligand concentrations. We found distinct populations of receptors as soon as 1 min after ligand stimulation, indicating early diversification of function. To understand this heterogeneity, we developed a mathematical model. The model predicted that variations in phosphorylation are dependent on the abundances of signaling partners, while phosphorylation levels are dependent on dimer lifetimes. The predictions were confirmed in studies of cell lines with different expression levels of signaling partners, and in experiments comparing low- and high-affinity ligands and oncogenic EGFR mutants. These results reveal how ligand-regulated receptor dimerization dynamics and adaptor protein concentrations play critical roles in EGFR signaling.

SUBMITTER: Salazar-Cavazos E

PROVIDER: S-EPMC7202077 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Multisite EGFR phosphorylation is regulated by adaptor protein abundances and dimer lifetimes.

Salazar-Cavazos Emanuel E Nitta Carolina Franco CF Mitra Eshan D ED Wilson Bridget S BS Lidke Keith A KA Hlavacek William S WS Lidke Diane S DS

Molecular biology of the cell 20200108 7

Differential epidermal growth factor receptor (EGFR) phosphorylation is thought to couple receptor activation to distinct signaling pathways. However, the molecular mechanisms responsible for biased signaling are unresolved due to a lack of insight into the phosphorylation patterns of full-length EGFR. We extended a single-molecule pull-down technique previously used to study protein-protein interactions to allow for robust measurement of receptor phosphorylation. We found that EGFR is predomina ...[more]

PMID: 31913761

Similar Datasets

Project description:Deregulation of epidermal growth factor receptor (EGFR) signaling has been correlated with the development of a variety of human carcinomas. EGF-induced receptor dimerization and consequent trans- auto-phosphorylation are among the earliest events in signal transduction. Binding of EGF is thought to induce a conformational change that consequently unfolds an ectodomain loop required for dimerization indirectly. It may also induce important allosteric changes in the cytoplasmic domain. Despite extensive knowledge on the physiological activation of EGFR, the effect of targeted therapies on receptor conformation is not known and this particular aspect of receptor function, which can potentially be influenced by drug treatment, may in part explain the heterogeneous clinical response among cancer patients. Here, we used Förster resonance energy transfer/fluorescence lifetime imaging microscopy (FRET/FLIM) combined with two-color single-molecule tracking to study the effect of ATP-competitive small molecule tyrosine kinase inhibitors (TKIs) and phosphatase-based manipulation of EGFR phosphorylation on live cells. The distribution of dimer on-times was fitted to a monoexponential to extract dimer off-rates (koff). Our data show that pretreatment with gefitinib (active conformation binder) stabilizes the EGFR ligand-bound homodimer. Overexpression of EGFR-specific DEP-1 phosphatase was also found to have a stabilizing effect on the homodimer. No significant difference in the koff of the dimer could be detected when an anti-EGFR antibody (425 Snap single-chain variable fragment) that allows for dimerization of ligand-bound receptors, but not phosphorylation, was used. These results suggest that both the conformation of the extracellular domain and phosphorylation status of the receptor are involved in modulating the stability of the dimer. The relative fractions of these two EGFR subpopulations (interacting versus free) were obtained by a fractional-intensity analysis of ensemble FRET/FLIM images. Our combined imaging approach showed that both the fraction and affinity (surrogate of conformation at a single-molecule level) increased after gefitinib pretreatment or DEP-1 phosphatase overexpression. Using an EGFR mutation (I706Q, V948R) that perturbs the ability of EGFR to dimerize intracellularly, we showed that a modest drug-induced increase in the fraction/stability of the EGFR homodimer may have a significant biological impact on the tumor cell's proliferation potential.

Dataset Information

Multisite EGFR phosphorylation is regulated by adaptor protein abundances and dimer lifetimes.

Publications

Multisite EGFR phosphorylation is regulated by adaptor protein abundances and dimer lifetimes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets