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Basic Amino Acids Within the Juxtamembrane Domain of the Epidermal Growth Factor Receptor Regulate Receptor Dimerization and Auto-phosphorylation

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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.

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Data Availability

The data, expression plasmids, and cell lines used in this study are available upon reasonable request.

Code Availability

Software application or custom code: not applicable.

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

DSS:

Disuccinimidyl suberate intracellular crosslinker

BS3 :

Bis(sulfosuccinimidyl)suberate extracellular crosslinker

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Acknowledgements

We thank Dr. Maurine Linder (Cornell University) for the use of her VersaDoc MP 5000 imaging system. We appreciate support of these studies by the National Institutes of Health (grants cited elsewhere); the content of this paper is solely the responsibility of the authors.

Funding

This study was supported by the National Institutes of Health (Grant Nos. R01 GM117552 and R01 AI022449).

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Authors and Affiliations

  1. Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA

    Jordan D. Mohr, Alice Wagenknecht-Wiesner, David A. Holowka & Barbara A. Baird

  2. Graduate Field of Pharmacology, Cornell University, Ithaca, NY, USA

    Jordan D. Mohr & Barbara A. Baird

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Correspondence to Barbara A. Baird.

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Online Resource 1

NIH 3T3 cells transiently expressing wt-EGFR are constitutively active. NIH 3T3 cells were transiently transfected with wt-EGFR or EGFR R1–6, EGF stimulated (or not) and chemically crosslinked with BS3, lysed, and immunoblotted for EGFR pY1173, total EGFR, and β-actin. Constitutive activation in the absence of EGF (lane 1) is seen in unstimulated cells expressing wt EGFR, likely due to overexpression. The appearance of double band for β-actin is probably due to nonspecific binding to another cell component of slightly higher molecular weight. Supplementary file 1 (TIF 12872 KB)

Online Resource 2

RBL-2H3 cells transiently expressing wt EGFR at low levels do not exhibit constitutive activation. RBL 2H3 cells were transiently transfected with wt-EGFR or EGFR R1–6, EGF stimulated (or not) and chemically crosslinked or not with BS3, lysed, and immunoblotted for EGFR pY1173, total EGFR, and β-actin. There is no constitutive activation in unstimulated cells expressing wt EGFR in the absence of EGF (lane 6); after addition of EGF phosphorylated dimers are trapped by chemical crosslinking (lanes 3-5). Supplementary file 2 (TIF 18105 KB)

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Mohr, J.D., Wagenknecht-Wiesner, A., Holowka, D.A. et al. Basic Amino Acids Within the Juxtamembrane Domain of the Epidermal Growth Factor Receptor Regulate Receptor Dimerization and Auto-phosphorylation. Protein J 39, 476–486 (2020). https://doi.org/10.1007/s10930-020-09943-8

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