The Journal of Membrane Biology

, Volume 252, Issue 4–5, pp 357–369 | Cite as

Modulation of Transmembrane Domain Interactions in Neu Receptor Tyrosine Kinase by Membrane Fluidity and Cholesterol

  • Muhammad Hasan
  • Dharmesh Patel
  • Natalie Ellis
  • Steven P. Brown
  • Józef R. Lewandowski
  • Ann M. DixonEmail author
Part of the following topical collections:
  1. Membrane and Receptor Dynamics


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.


Neu 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

Supplementary material

232_2019_75_MOESM1_ESM.docx (4.5 mb)
Supplementary material 1 (DOCX 4647 kb)


  1. Abdine A, Verhoeven MA, Park K-H et al (2010) Structural study of the membrane protein MscL using cell-free expression and solid-state NMR. J Magn Reson 204:155–159. CrossRefPubMedGoogle Scholar
  2. Alves AC, Ribeiro D, Nunes C, Reis S (2016) Biophysics in cancer: the relevance of drug-membrane interaction studies. Biochim Biophys Acta 1858:2231–2244. CrossRefPubMedGoogle Scholar
  3. Anbazhagan V, Munz C, Tome L, Schneider D (2010) Fluidizing the membrane by a local anesthetic: phenylethanol affects membrane protein oligomerization. J Mol Biol 404:773–777. CrossRefPubMedGoogle Scholar
  4. Baier CJ, Fantini J, Barrantes FJ (2011) Disclosure of cholesterol recognition motifs in transmembrane domains of the human nicotinic acetylcholine receptor. Sci Rep 1:69. CrossRefPubMedPubMedCentralGoogle Scholar
  5. Bargmann CI, Weinberg RA (1988a) Oncogenic activation of the neu-encoded receptor protein by point mutation and deletion. EMBO J 7:2043–2052. CrossRefPubMedPubMedCentralGoogle Scholar
  6. Bargmann CI, Weinberg RA (1988b) Increased tyrosine kinase activity associated with the protein encoded by the activated neu oncogene. Proc Natl Acad Sci USA 85:5394–5398. CrossRefPubMedGoogle Scholar
  7. Bargmann CI, Hung M-C, Weinberg RA (1986) Multiple independent activations of the neu oncogene by a point mutation altering the transmembrane domain of p185. Cell 45:649–657. CrossRefPubMedGoogle Scholar
  8. Beevers AJ, Kukol A (2006) The transmembrane domain of the oncogenic mutant ErbB-2 receptor: a structure obtained from site-specific infrared dichroism and molecular dynamics. J Mol Biol 361:945–953. CrossRefPubMedGoogle Scholar
  9. Beevers AJ, Damianoglou A, Oates J et al (2010) Sequence-dependent oligomerization of the neu transmembrane domain suggests inhibition of “conformational switching” by an oncogenic mutant. Biochemistry 49:2811–2820. CrossRefPubMedGoogle Scholar
  10. 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. CrossRefGoogle Scholar
  11. Bell CA, Tynan JA, Hart KC et al (2000) Rotational coupling of the transmembrane and kinase domains of the Neu receptor tyrosine kinase. Mol Biol Cell 11:3589–3599. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Bennett AE, Rienstra CM, Auger M, Lakshmi KV, Griffin RG (1995) Heteronuclear decoupling in rotating solids. J Chem Phys 103:6951–6958. CrossRefGoogle Scholar
  13. Bezrukov SM (2000) Functional consequences of lipid packing stress. Curr Opin Colloid Interface Sci 5:237–243. CrossRefGoogle Scholar
  14. Bocharov EV, Mineev KS, Volynsky PE et al (2008) Spatial structure of the dimeric transmembrane domain of the growth factor receptor ErbB2 presumably corresponding to the receptor active state. J Biol Chem 283:6950–6956. CrossRefPubMedGoogle Scholar
  15. Boughter CT, Monje-Galvan V, Im W, Klauda JB (2016) Influence of cholesterol on phospholipid bilayer structure and dynamics. J Phys Chem B 120:11761–11772. CrossRefPubMedGoogle Scholar
  16. Bublil EM, Yarden Y (2007) The EGF receptor family: spearheading a merger of signaling and therapeutics. Curr Opin Cell Biol 19:124–134. CrossRefPubMedGoogle Scholar
  17. 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. CrossRefPubMedGoogle Scholar
  18. Cascio M, Wallace BA (1995) Effects of local environment on the circular dichroism spectra of polypeptides. Anal Biochem 227:90–100. CrossRefPubMedGoogle Scholar
  19. Chandra P, Noh H-B, Shim Y-B (2013) Cancer cell detection based on the interaction between an anticancer drug and cell membrane components. Chem Commun 49:1900–1902. CrossRefGoogle Scholar
  20. 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. CrossRefPubMedGoogle Scholar
  21. Cooper RA (1978) Influence of increased membrane cholesterol on membrane fluidity and cell function in human red blood cells. J Supramol Struct 8:413–430. CrossRefPubMedGoogle Scholar
  22. de Kruijff B (1997) Lipid polymorphism and biomembrane function. Curr Opin Chem Biol 1:564–569. CrossRefPubMedGoogle Scholar
  23. De Meyer FJM, Benjamini A, Rodgers JM et al (2010) Molecular simulation of the DMPC-cholesterol phase diagram. J Phys Chem B. 114:10451–10461. CrossRefPubMedGoogle Scholar
  24. Dell’Era Dosch D, Ballmer-Hofer K (2010) Transmembrane domain-mediated orientation of receptor monomers in active VEGFR-2 dimers. FASEB J 24:32–38. CrossRefGoogle Scholar
  25. Dergunov AD, Savushkin EV, Dergunova LV, Litvinov DY (2019) Significance of cholesterol-binding motifs in ABCA1, ABCG1, and SR-B1 Structure. J Membr Biol 252:41–60. CrossRefPubMedGoogle Scholar
  26. Endres NF, Das R, Smith AW et al (2013) Conformational coupling across the plasma membrane in activation of the EGF receptor. Cell 152:543–556. CrossRefPubMedPubMedCentralGoogle Scholar
  27. Epand RM (1998) Lipid polymorphism and protein–lipid interactions. Biochim Biophys Acta 1376:353–368. CrossRefPubMedGoogle Scholar
  28. Epand RM (2006) Cholesterol and the interaction of proteins with membrane domains. Prog Lipid Res 45:279–294. CrossRefPubMedGoogle Scholar
  29. Escriba PV, Martin ML, Noguera-Salva MA et al (2011) Sphingomyelin and sphingomyelin synthase (SMS) in the malignant transformation of glioma cells and in 2-hydroxyoleic acid therapy. Proc Natl Acad Sci 108:19569–19574. CrossRefPubMedGoogle Scholar
  30. Fantini J, Barrantes FJ (2013) How cholesterol interacts with membrane proteins: an exploration of cholesterol-binding sites including CRAC, CARC, and tilted domains. Front Physiol 4:31. CrossRefPubMedPubMedCentralGoogle Scholar
  31. Feinstein MB, Fernandez SM, Sha’afi RI (1975) Fluidity of natural membranes and phosphatidylserine and ganglioside dispersions. Biochim Biophys Acta 413:354–370. CrossRefPubMedGoogle Scholar
  32. Fox TE, Young MM, Pedersen MM et al (2011) Insulin signaling in retinal neurons is regulated within cholesterol-enriched membrane microdomains. Am J Physiol Metab 300:E600–E609. CrossRefGoogle Scholar
  33. Frericks HL, Zhou DH, Yap LL et al (2006) Magic-angle spinning solid-state NMR of a 144 kDa membrane protein complex: E. coli cytochrome bo3 oxidase. J Biomol NMR 36:55–71. CrossRefPubMedGoogle Scholar
  34. Fung BM, Khitrin AK, Ermolaev K (2000) An Improved broadband decoupling sequence for liquid crystals and solids. J Magn Reson 142:97–101. CrossRefPubMedGoogle Scholar
  35. 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. CrossRefPubMedGoogle Scholar
  36. Ge G, Wu J, Lin Q (2001) Effect of membrane fluidity on tyrosine kinase activity of reconstituted epidermal growth factor receptor. Biochem Biophys Res Commun 282:511–514. CrossRefPubMedGoogle Scholar
  37. Goddard Td, Kneller DG (2004) SPARKY 3. Univ California, San Fr. CrossRefGoogle Scholar
  38. Hiller M, Krabben L, Vinothkumar KR et al (2005) Solid-state magic-angle spinning NMR of outer-membrane protein G from Escherichia coli. ChemBioChem 6:1679–1684. CrossRefPubMedGoogle Scholar
  39. Holbro T, Beerli RR, Maurer F et al (2003) The ErbB2/ErbB3 heterodimer functions as an oncogenic unit: ErbB2 requires ErbB3 to drive breast tumor cell proliferation. Proc Natl Acad Sci 100:8933–8938. CrossRefPubMedGoogle Scholar
  40. Houliston RS, Hodges RS, Sharom FJ, Davis JH (2004) Characterization of the proto-oncogenic and mutant forms of the transmembrane region of Neu in micelles. J Biol Chem 279:24073–24080. CrossRefPubMedGoogle Scholar
  41. Jaipuria G, Leonov A, Giller K et al (2017) Cholesterol-mediated allosteric regulation of the mitochondrial translocator protein structure. Nat Commun 8:14893. CrossRefPubMedPubMedCentralGoogle Scholar
  42. Johnson SM, Robinson R (1979) The composition and fluidity of normal and leukaemic or lymphomatous lymphocyte plasma membranes in mouse and man. Biochim Biophys Acta 558:282–295. CrossRefPubMedGoogle Scholar
  43. Jones DH, Barber KR, Grant CW (1998) Sequence-related behaviour of transmembrane domains from class I receptor tyrosine kinases. Biochim Biophys Acta 1371:199–212. CrossRefPubMedGoogle Scholar
  44. Khemtémourian L, Buchoux S, Aussenac F, Dufourc EJ (2007) Dimerization of Neu/Erb2 transmembrane domain is controlled by membrane curvature. Eur Biophys J 36:107–112. CrossRefPubMedGoogle Scholar
  45. Killian JA (1998) Hydrophobic mismatch between proteins and lipids in membranes. Biochim Biophys Acta 1376:401–416. CrossRefPubMedGoogle Scholar
  46. Kučerka N, Pencer J, Nieh MP, Katsaras J (2007) Influence of cholesterol on the bilayer properties of monounsaturated phosphatidylcholine unilamellar vesicles. Eur Phys J 23:247–254. CrossRefGoogle Scholar
  47. Labrecque L, Royal I, Surprenant DS et al (2003) Regulation of vascular endothelial growth factor receptor-2 activity by Caveolin-1 and plasma membrane cholesterol. Mol Biol Cell 14:334–347. CrossRefPubMedPubMedCentralGoogle Scholar
  48. Lemmon MA, Schlessinger J (2010) Cell signalling by receptor tyrosine kinases. Cell 141:1117–1134. CrossRefPubMedPubMedCentralGoogle Scholar
  49. 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. CrossRefPubMedGoogle Scholar
  50. Lindon AH, Franks WT, Akbey Ü, Lange S et al (2011) Cryogenic temperature effects and resolution upon slow cooling of protein preparations in solid state NMR. J Biomol NMR 51:283–292. CrossRefGoogle Scholar
  51. Lund-Katz S, Laboda HM, McLean LR, Phillips MC (1988) Influence of molecular packing and phospholipid type on rates of cholesterol exchange. Biochemistry 27:3416–3423. CrossRefPubMedGoogle Scholar
  52. Markley JL, Ulrich EL, Berman HM et al (2008) BioMagResBank (BMRB) as a partner in the worldwide protein data bank (wwPDB): new policies affecting biomolecular NMR depositions. J Biomol NMR 40:153–155. CrossRefPubMedPubMedCentralGoogle Scholar
  53. Maruyama IN (2015) Activation of transmembrane cell-surface receptors via a common mechanism? The “rotation model”. BioEssays 37:959–967. CrossRefPubMedPubMedCentralGoogle Scholar
  54. Maurya SR, Chaturvedi D, Mahalakshmi R (2013) Modulating lipid dynamics and membrane fluidity to drive rapid folding of a transmembrane barrel. Sci Rep 3:1989. CrossRefPubMedPubMedCentralGoogle Scholar
  55. Metz G, Wu X, Smith SO (1994) Ramped-amplitude cross polarization in magic-angle-spinning NMR. J Magn Reson Ser A 110:219–227. CrossRefGoogle Scholar
  56. Mitri Z, Constantine T, O’Regan R (2012) The HER2 receptor in breast cancer: pathophysiology, clinical use, and new advances in therapy. Chemother Res Pract 2012:1–7. CrossRefGoogle Scholar
  57. Moriki T, Maruyama H, Maruyama IN (2001) Activation of preformed EGF receptor dimers by ligand-induced rotation of the transmembrane domain. J Mol Biol 311:1011–1026. CrossRefPubMedGoogle Scholar
  58. Needham D, McIntosh TJ, Evans E (1988) Thermomechanical and transition properties of dimyristoylphosphatidylcholine/cholesterol bilayers. Biochemistry 27:4668–4673. CrossRefPubMedGoogle Scholar
  59. Nomura K, Lintuluoto M, Morigaki K (2011) Hydration and temperature dependence of 13C and 1H NMR spectra of the DMPC phospholipid membrane and complete resonance assignment of its crystalline state. J Phys Chem B 115:14991–15001. CrossRefPubMedGoogle Scholar
  60. Paschkowsky S, Recinto SJ, Young JC et al (2018) Membrane cholesterol as regulator of human rhomboid protease RHBDL4. J Biol Chem 293:15556–15568. CrossRefPubMedPubMedCentralGoogle Scholar
  61. Pike LJ, Casey L (2002) Cholesterol levels modulate EGF receptor-mediated signalling by altering receptor function and trafficking. Biochemistry 41:10315–10322. CrossRefPubMedGoogle Scholar
  62. Prakash A, Janosi L, Doxastakis M (2011) GxxxG motifs, phenylalanine, and cholesterol guide the self-association of transmembrane domains of ErbB2 receptors. Biophys J 101:1949–1958. CrossRefPubMedPubMedCentralGoogle Scholar
  63. Purba E, Saita E, Maruyama I (2017) Activation of the EGF receptor by ligand binding and oncogenic mutations: the “Rotation model”. Cells 6:13. CrossRefPubMedCentralGoogle Scholar
  64. Rance M, Byrd RA (1983) Obtaining high-fidelity spin-1/2 powder spectra in anisotropic media: phase-cycled Hahn echo spectroscopy. J Magn Reson 52:221–240. CrossRefGoogle Scholar
  65. Russ WP, Engelman DM (2000) The GxxxG motif: a framework for transmembrane helix-helix association. J Mol Biol 296:911–919. CrossRefPubMedGoogle Scholar
  66. Schlessinger J (2002) Ligand-induced, receptor-mediated dimerization and activation of EGF receptor. Cell 110:669–672. CrossRefPubMedGoogle Scholar
  67. Schuh JR, Banerjee U, Müller L, Chan SI (1982) The phospholipid packing arrangement in small bilayer vesicles as revealed by proton magnetic resonance studies at 500 MHz. Biochim Biophys Acta 687:219–225. CrossRefPubMedGoogle Scholar
  68. Sengupta D, Chattopadhyay A (2012) Identification of cholesterol binding sites in the Serotonin 1A receptor. J Phys Chem B 116:12991–12996. CrossRefPubMedGoogle Scholar
  69. Serra V, Vivancos A, Puente XS et al (2013) Clinical response to a Lapatinib-based therapy for a Li-Fraumeni syndrome patient with a novel HER2 V659E mutation. Cancer Discov 3:1238–1244. CrossRefPubMedGoogle Scholar
  70. Shinitzky M (1984) Membrane fluidity in malignancy adversative and recuperative. Biochim Biophys Acta 738:251–261. CrossRefPubMedGoogle Scholar
  71. Simons K (2000) How cells handle cholesterol. Science 290:1721–1726. CrossRefPubMedGoogle Scholar
  72. Smith SO, Smith CS, Bormann BJ (1996) Strong hydrogen bonding interactions involving a buried glutamic acid in the transmembrane sequence of the neu/erbB-2 receptor. Nat Struct Biol 3:252–258. CrossRefPubMedGoogle Scholar
  73. Smith SO, Smith C, Shekar S et al (2002) Transmembrane interactions in the activation of the Neu receptor tyrosine kinase. Biochemistry 41:9321–9332. CrossRefPubMedGoogle Scholar
  74. Sperotto MM, Mouritsen OG (1988) Dependence of lipid membrane phase transition temperature on the mismatch of protein and lipid hydrophobic thickness. Eur Biophys J 16:1–10. CrossRefGoogle Scholar
  75. Sternberg MJE, Gullick WJ (1990) A sequence motif in the transmembrane region of growth factor receptors with tyrosine kinase activity mediates dimerization. Protein Eng 3:245–248. CrossRefPubMedGoogle Scholar
  76. Strandberg E, Killian JA (2003) Snorkeling of lysine side chains in transmembrane helices: how easy can it get? FEBS Lett 544:69–73. CrossRefPubMedGoogle Scholar
  77. Taghibiglou C, Bradley CA, Gaertner T et al (2009) Mechanisms involved in cholesterol-induced neuronal insulin resistance. Neuropharmacology 57:268–276. CrossRefPubMedGoogle Scholar
  78. Takegoshi K, Terao T (2002) 13C nuclear Overhauser polarization nuclear magnetic resonance in rotating solids: replacement of cross polarization in uniformly 13C labeled molecules with methyl groups. J Chem Phys 117:1700–1707. CrossRefGoogle Scholar
  79. 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. CrossRefPubMedGoogle Scholar
  80. Tan M, Yu D (2007) Molecular mechanisms of ErbB2-mediated breast cancer chemoresistance. In: Yu D, Hung MC (eds) Breast Cancer Chemosensitivity. Advances in Experimental Medicine and Biology, vol 608. Springer, New York, pp 119–129CrossRefGoogle Scholar
  81. Tao R-H, Maruyama IN (2008) All EGF(ErbB) receptors have preformed homo- and heterodimeric structures in living cells. J Cell Sci 121:3207–3217. CrossRefPubMedGoogle Scholar
  82. Taraboletti G, Perin L, Bottazzi B et al (1989) Membrane fluidity affects tumor-cell motility, invasion and lung-colonizing potential. Int J Cancer 44:707–713. CrossRefPubMedGoogle Scholar
  83. Vist MR, Davis JH (1990) Phase equilibria of cholesterol/dipalmitoylphosphatidylcholine mixtures: deuterium nuclear magnetic resonance and differential scanning calorimetry. Biochemistry 29:451–464. CrossRefPubMedGoogle Scholar
  84. Wallace BA (2003) Analyses of circular dichroism spectra of membrane proteins. Protein Sci 12:875–884. CrossRefPubMedPubMedCentralGoogle Scholar
  85. Wang R, Zhang Y, Pan Y et al (2015) Comprehensive investigation of oncogenic driver mutations in Chinese non-small cell lung cancer patients. Oncotarget 6:34300–34308. CrossRefPubMedPubMedCentralGoogle Scholar
  86. Weiner DB, Liu J, Cohen JA et al (1989) A point mutation in the neu oncogene mimics ligand induction of receptor aggregation. Nature 339:230–231. CrossRefPubMedGoogle Scholar
  87. Yamamoto H, Higasa K, Sakaguchi M et al (2014) Novel germline mutation in the transmembrane domain of HER2 in familial lung adenocarcinomas. J Natl Cancer Inst 106:djt338. CrossRefPubMedGoogle Scholar
  88. Yarden Y, Schlessinger J (1987) Epidermal growth factor induces rapid, reversible aggregation of the purified epidermal growth factor receptor. Biochemistry 26:1443–1451. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of ChemistryUniversity of WarwickCoventryUK
  2. 2.Department of PhysicsUniversity of WarwickCoventryUK

Personalised recommendations