Basic Amino Acids Within the Juxtamembrane Domain of the Epidermal Growth Factor Receptor Regulate Receptor Dimerization
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Basic Amino Acids Within the Juxtamembrane Domain of the Epidermal Growth Factor Receptor Regulate Receptor Dimerization and Auto‑phosphorylation Jordan D. Mohr1,2 · Alice Wagenknecht‑Wiesner1 · David A. Holowka1 · Barbara A. Baird1,2 Accepted: 10 November 2020 / Published online: 19 November 2020 © Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract Epidermal growth factor receptor (EGFR) dysregulation is observed in many human cancers and is both a cause of oncogenesis and a target for chemotherapy. We previously showed that partial charge neutralization of the juxtamembrane (JX) region of EGFR via the EGFR R1–6 mutant construct induces constitutive receptor activation and transformation of NIH 3T3 cells, both from the plasma membrane and from the ER when combined with the ER-retaining L417H mutation (Bryant et al. in J Biol Chem 288:34930–34942, 2013). Here, we use chemical crosslinking and immunoblotting to show that these mutant constructs form constitutive, phosphorylated dimers in both the plasma membrane and the ER. Furthermore, we combine this electrostatic perturbation with conformationally-restricted receptor mutants to provide evidence that activation of EGFR R1–6 dimers requires functional coupling both between the EGFR extracellular dimerization arms and between intracellular tyrosine kinase domains. These findings provide evidence that the electrostatic charge of the JX region normally serves as a negative regulator of functional dimerization of EGFR. Keywords EGFR · Juxtamembrane region · Electrostatic regulation · Dimerization Abbreviations EGFR Epidermal growth factor receptor ECD Extracellular domain JX Juxtamembrane TKD Tyrosine kinase domain RTK Receptor tyrosine kinase EGFR R1–6 EGFR JX charge-reduced mutant EGFR L417H ER-retained EGFR mutant EGFR 246–253* EGFR ECD domain III mutant that cannot form ECD dimers EGFR V948R EGFR TKD mutant that cannot form asymmetric TKD dimers
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10930-020-09943-8) contains supplementary material, which is available to authorized users. * Barbara A. Baird [email protected] 1
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA
Graduate Field of Pharmacology, Cornell University, Ithaca, NY, USA
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DSS Disuccinimidyl suberate intracellular crosslinker BS3 Bis(sulfosuccinimidyl)suberate extracellular crosslinker
1 Introduction The epidermal growth factor receptor (EGFR or ErbB1) has been extensively examined as a receptor tyrosine kinase that controls a system of tightly regulated pro-survival signaling pathways within the cell [1–3]. Binding of ligand EGF causes dimerization of monomeric receptors and concomitant conversion to an active state, or possibly converts inactive, weakly dimerized receptors into an active state. Numerous mutations in EGFR cause spontaneous receptor activation and have been observed in human cancers [4]. As exemplified by the L834R mutant, oncog
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