Modulation of Transmembrane Domain Interactions in Neu Receptor Tyrosine Kinase by Membrane Fluidity and Cholesterol
- 168 Downloads
The activation mechanism of the ErbB family of receptors is of considerable medical interest as they are linked to a number of human cancers, including an aggressive form of breast cancer. In the rat analogue of the human ErbB2 receptor, referred to as Neu, a point mutation in the transmembrane domain (V664E) has been shown to trigger oncogenic transformation. While the structural impact of this mutation has been widely studied in the past to yield models for the active state of the Neu receptor, little is known about the impact of cholesterol on its structure. Given previous reports of the influence of cholesterol on other receptor tyrosine kinases (RTKs), as well as the modulation of lipid composition in cancer cells, we wished to investigate how cholesterol content impacts the structure of the Neu transmembrane domain. We utilized high-resolution magic angle spinning solid-state NMR to measure 13C–13C coupling of selectively labelled probe residues in the Neu transmembrane domain in lipid bilayers containing cholesterol. We observe inter-helical coupling between residues that support helix–helix interactions on both dimerization motifs reported in the literature (A661-XXX-G665 and I659-XXX-V663). We further explore how changes in cholesterol concentration alter transmembrane domain interactions and the properties and mechanics of the bilayer. We interpret our results in light of previous studies relating RTK activity to cholesterol enrichment and/or depletion, and propose a novel model to explain our data that includes the recognition and binding of cholesterol by the Neu transmembrane domain through a putative cholesterol-recognition/interaction amino acid consensus sequence.
KeywordsNeu oncogene Receptor tyrosine kinase Cholesterol-recognition Solid-state NMR Membrane bilayers
The authors would like to thank the EPSRC for provision of a PhD studentship through the MOAC Doctoral Training Centre (Grant Number EP/F500378/1) to M.H. and a PhD studentship through the Doctoral Training Partnership grant to D.P. The authors would also like to thank the BBSRC for provision of a PhD studentship through the BBSRC Doctoral Training Grant (BB/D52700X/1) to N.E. The authors wish to thank Jonathan M. Lamley (University of Warwick, Coventry, UK) for ssNMR assistance. The experimental data for this study are provided as a supporting data set from WRAP, the Warwick Research Archive Portal at http://wrap.warwick.ac.uk.
- Bargmann CI, Weinberg RA (1988a) Oncogenic activation of the neu-encoded receptor protein by point mutation and deletion. EMBO J 7:2043–2052. https://doi.org/10.1002/j.1460-2075.1988.tb03044.x CrossRefPubMedPubMedCentralGoogle Scholar
- Beevers AJ, Nash A, Salazar-Cancino M et al (2012) Effects of the oncogenic V664E mutation on membrane insertion, structure, and sequence-dependent interactions of the neu transmembrane domain in micelles and model membranes: an integrated biophysical and simulation study. Biochemistry 12:2558–2568. https://doi.org/10.1021/bi201269w CrossRefGoogle Scholar
- Cady SD, Mishanina TV, Hong M (2009) Structure of amantadine-bound M2 transmembrane peptide of Influenza A in lipid bilayers from magic-angle-spinning solid-state NMR: the role of Ser31 in amantadine binding. J Mol Biol 385:1127–1141. https://doi.org/10.1016/j.jmb.2008.11.022 CrossRefPubMedGoogle Scholar
- Chen Y, Wallace BA (1997) Secondary solvent effects on the circular dichroism spectra of polypeptides in non-aqueous environments: influence of polarisation effects on the far ultraviolet spectra of alamethicin. Biophys Chem 65:65–74. https://doi.org/10.1016/S0301-4622(96)02225-9 CrossRefPubMedGoogle Scholar
- Galeotti T, Borrello S, Minotti G, Masotti L (1986) Membrane alterations in cancer cells: the role of oxy radicals. Ann NY Acad Sci 488:468–480. https://doi.org/10.1111/j.1749-6632.1986.tb54425.x CrossRefPubMedGoogle Scholar
- Li H, Papadopoulos V (1998) Peripheral-type benzodiazepine receptor function in cholesterol transport. Identification of a putative cholesterol recognition/interaction amino acid sequence and consensus pattern. Endocrinology 139:4991–4997. https://doi.org/10.1210/endo.139.12.6390 CrossRefPubMedGoogle Scholar
- Takegoshi K, Imaizumi T, Terao T (2000) One- and two-dimensional 13C–1H/15N–1H dipolar correlation experiments under fast magic-angle spinning for determining the peptide dihedral angle φ. Solid State Nucl Magn Reson 16:271–278. https://doi.org/10.1016/S0926-2040(00)00076-X CrossRefPubMedGoogle Scholar