The EphA2 receptor is activated through induction of distinct, ligand-dependent oligomeric structures

Abstract

The EphA2 receptor tyrosine kinase is capable of activating multiple diverse signaling pathways with roles in processes such as tissue homeostasis and cancer. EphA2 is known to form activated oligomers in the presence of ephrin-A ligands. Here, we characterize the lateral interactions between full-length EphA2 molecules in the plasma membrane in the presence of three types of ligands (dimeric ephrinA1-Fc, monomeric ephrinA1, and an engineered peptide ligand) as well as in the absence of ligand, using a quantitative FRET technique. The data show that EphA2 forms higher-order oligomers and two different types of dimers that all lead to increased EphA2 tyrosine phosphorylation, which is indicative of increased kinase-dependent signaling. We find that different ligands stabilize conformationally distinct oligomers that are assembled through two different interfaces. Our results suggest that these different oligomeric assemblies could have distinct signaling properties, contributing to the diverse activities of the EphA2 receptor.

Dectin-1 Molecular Aggregation and Signaling is Sensitive to β-Glucan Structure and Glucan Exposure on Candida albicans Cell Walls

“…To accomplish this, we first employed Fluorescence-Lifetime Imaging Microscopy for Forster Resonance Energy Transfer (FLIM-FRET). FRET based imaging capitalizes on close proximity of two proteins to visualize protein-protein interactions, including receptor dimerization and receptor-ligand complex formation [60]. FLIM characterizes the duration of a fluorophore’s excited state before returning to the ground state.…”

Engineering nanomolar peptide ligands that differentially modulate EphA2 receptor signaling

“…Using a quantitative FRET approach in live cells, we have previously shown that in HEK293 cells transiently transfected with EphA2, the receptor weakly dimerizes in the absence of a bound ligand (22) through an extracellular interface known as the "clustering" interface (19,20). Furthermore, we found that the YSA-GSGSK (3) peptide increases the formation of these EphA2 unliganded dimers (23,24). In contrast, the monomeric , total EphA2 levels, and AKT phosphorylation on Ser 473 (pAKT, indicative of AKT activation).…”

“…In these experiments, FRET was measured in 300 -400 individual cells with different EphA2 expression levels. FRET efficiency, EphA2-mTURQ (donor) concentration, and EphA2-EYFP (acceptor) concentration in the plasma membrane were measured in micrometer-sized regions of the plasma membrane as described (23), and the data derived from the individual cells were combined to yield binding curves. A least-square error analysis of the FRET data, performed as described previously (25), showed that these data are best described by a monomer-dimer association model rather than a higher-order association model.…”

The Ephrin receptor A2 and Roundabout Guidance Receptor 1 heterodimer: A potential theranostic for squamous cell carcinomas

“…Upon binding to their ligands, they often oligomerize and crossphosphorylate each other, especially on tyrosine residues at the critical juxtamembrane domain and at the activation loop (49-51) to transduce various cellular signals. In the case of the EPH receptors, many of which are upregulated in numerous cancers, ligand activation leads to inhibition of tumor growth, migration and invasiveness (51). In our study when SCC cells were treated with EphrinA1 the natural ligand of EPHA2 (non-tumorigenic signaling) in in vitro culture, we did not see an inhibition in cellular proliferation of LSCC and HNSCC cells underscoring the need to study the presence of other degenerate unexplored pathways regulating EPHA2 signaling.…”