Lipid-protein interplay in dimerization of the juxtamembrane domains of epidermal growth factor receptor

Abstract

Transmembrane (TM) helix and juxtamembrane (JM) domains (TM-JM) bridge the extracellular and intracellular domains of single-pass membrane proteins, including epidermal growth factor receptor (EGFR). TM-JM dimerization plays a crucial role in regulation of EGFR kinase activity at the cytoplasmic side. Although the interaction of JM with membrane lipids is thought to be important to turn on EGF signaling and phosphorylation of Thr654 on JM leads to desensitization, the underlying kinetic mechanisms remain unclear. Especially, how Thr654 phosphorylation regulates EGFR activity is largely unknown. Here, combining single-pair FRET imaging and nanodisc techniques, we showed that phosphatidylinositol 4,5-bis phosphate (PIP2) facilitated JM dimerization effectively. We also found that Thr654 phosphorylation dissociated JM dimers in the membranes containing acidic lipids, suggesting that Thr654 phosphorylation electrostatically prevented the interaction with basic residues in JM and acidic lipids. Based on the single-molecule experiment, we clarified the kinetic pathways of monomer (inactive state) -dimer (active state) transition of JM domains and alteration in the pathways depending on the membrane lipid species and Thr654 phosphorylation.. CC-BY-NC-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was . http://dx.doi.org/10.1101/197673 doi: bioRxiv preprint first posted online Oct. 19, 2017; 3 Epidermal growth factor receptor (EGFR), the best studied receptor tyrosine kinase (RTK), plays an important role in regulating cell proliferation and differentiation 1,2 .EGFR consists of an extracellular domain that interacts with extracellular ligands, a single-pass transmembrane (TM) helix, a juxtamembrane (JM) domain, a cytoplasmic kinase domain that activates various signal cascades in the cell, and a C-terminal tail domain 3,4 . When ligands such as EGF bind to the extracellular domain of EGFR, conformational changes occur in the extracellular domain and the asymmetric dimer of intracellular kinase domains is formed 5 . This asymmetric dimerization enables one (activator) kinase domain to activate the other (receiver) kinase domain 6 , resulting in subsequent phosphorylation of tyrosine residues on the C-terminal tails of the activator and recruitment of intracellular signal proteins containing an SH2 and/or PTB domain, such as Shc, Grb2, etc. 7 .Although the structures of most of the EGFR domains excluding the C-tail domain 5,6,[8][9][10] have been solved individually, the overall architecture remains unclear. In particular, the most important question regarding the regulation of EGFR kinase activity across the lipid bilayer, i.e., the correlation between conformational changes in the extracellular domain upon ligand binding and rearrangement of the intracellular kinase domain, is still controversial.In the context of transmission of information across the membrane, the single-pass TM helix and the JM domain (TM-JM...

Bilateral regulation of EGFR activity and local PI dynamics observed with superresolution microscopy

“…7E, right). We have reported previously that Thr654 phosphorylation causes the JM-A dimer to dissociate in the presence of acidic lipids (Maeda et al, 2018;Maeda et al, 2022). This constitutes the negative feedback regulation of the EGFR kinase function.…”

“…In silico and in vitro analyses have suggested that PI(4,5)P 2 is involved in the dimerization of EGFR (Abd Halim et al, 2015;Maeda et al, 2018;Maeda et al, 2022;Matsushita et al, 2013). The aggregation of PI(4,5)P 2 with EGFR in the plasma membrane may help to stabilize the dimers of EGFR after its association with EGF.…”

“…Multiscale molecular dynamic simulations have suggested that phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 ) interacts specifically with the basic residues in the JM-A region, stabilizing the JM-A helices in an orientation away from the membrane surface by binding to PI(4,5)P 2 (Abd Halim et al, 2015;Matsushita et al, 2013). In addition to these in silico analyses, our recent in vitro study using nanodisc techniques suggested that phosphatidylserine (PS) and PI(4,5)P 2 stabilize the conformation of the JM-A dimer (Maeda et al, 2018;Maeda et al, 2022). Apart from these in silico and in vitro studies, the localization and roles of PS and PI(4,5)P 2 during EGFR activation have not been fully investigated experimentally in living cells due to the difficulty of visualizing the local lipid domains around EGFR in the plasma membrane.…”