Rybak, J. A. | Search bioRxiv

bioRxiv2024

Phosphatidylinositol (4,5)-bisphosphate drives the formation of EGFR and EphA2 complexes

Receptor tyrosine kinases (RTKs) regulate many cellular functions and are important targets in pharmaceutical development, particularly in cancer treatment. EGFR and EphA2 are two key RTKs that are associated with oncogenic phenotypes. Several studies have reported functional interplay between these receptors, but the mechanism of interaction is still unresolved. Here we utilize a time-resolved fluorescence spectroscopy called PIE-FCCS to resolve EGFR and EphA2 interactions in live cells. We tested the role of ligands and found that EGF, but not ephrin A1 (EA1), stimulated hetero-multimerization between the receptors. To determine the effect of anionic lipids, we targeted phospholipase C (PLC) activity to alter the abundance of phosphatidylinositol (4,5)-bisphosphate (PIP2). We found that higher PIP2 levels increased homo-multimerization of both EGFR and EphA2, as well as hetero-multimerization. This study provides a direct characterization of EGFR and EphA2 interactions in live cells and shows that PIP2 can have a substantial effect on the spatial organization of RTKs.

biophysics↗

bioRxiv2022

Allosteric inhibition of the epidermal growth factor receptor through disruption of transmembrane interactions

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK) commonly targeted for inhibition by anti-cancer therapeutics. Current therapeutics target EGFRs kinase domain or extracellular region. However, these types of inhibitors are not specific for tumors over healthy tissue and therefore cause undesirable side effects. Our lab has recently developed a new strategy to regulate RTK activity by designing a peptide that specifically binds to the transmembrane (TM) region of the RTK to allosterically modify kinase activity. These peptides are acidity-responsive, allowing them to preferentially target acidic environments like tumors. We have applied this strategy to EGFR and created the PET1 peptide. We observed that PET1 behaves as a pH-responsive peptide that modulates the configuration of the EGFR TM through a direct interaction. Our data indicated that PET1 inhibits EGFR-mediated cell migration. Finally, we investigated the mechanism of inhibition through molecular dynamics simulations, which showed that PET1 sits between the two EGFR TM helices; this molecular mechanism was additionally supported by AlphaFold-Multimer predictions. We propose that the PET1-induced disruption of native TM interactions disturbs the conformation of the kinase domain in such a way that it inhibits EGFRs ability to send migratory cell signals. This study is a proof-of-concept that acidity-responsive membrane peptide ligands can be generally applied to RTKs. In addition, PET1 constitutes a viable approach to therapeutically target the TM of EGFR.

cell biology↗