Journal of Membrane Biology

, Volume 215, Issue 2–3, pp 93–103 | Cite as

Studies of Receptor Tyrosine Kinase Transmembrane Domain Interactions: The EmEx-FRET Method

  • Mikhail Merzlyakov
  • Lirong Chen
  • Kalina Hristova


The energetics of transmembrane (TM) helix dimerization in membranes and the thermodynamic principles behind receptor tyrosine kinase (RTK) TM domain interactions during signal transduction can be studied using Förster resonance energy transfer (FRET). For instance, FRET studies have yielded the stabilities of wild-type fibroblast growth factor receptor 3 (FGFR3) TM domains and two FGFR3 pathogenic mutants, Ala391Glu and Gly380Arg, in the native bilayer environment. To further our understanding of the molecular mechanisms of deregulated FGFR3 signaling underlying different pathologies, we determined the effect of the Gly382Asp FGFR3 mutation, identified in a multiple myeloma cell line, on the energetics of FGFR3 TM domain dimerization. We measured dimerization energetics using a novel FRET acquisition and processing method, termed “emission-excitation FRET (EmEx-FRET),” which improves the precision of thermodynamic measurements of TM helix association. The EmEx-FRET method, verified here by analyzing previously published data for wild-type FGFR3 TM domain, should have broad utility in studies of protein interactions, particularly in cases when the concentrations of fluorophore-tagged molecules cannot be controlled.


Receptor tyrosine kinase Transmembrane domain FRET 



We thank our colleagues Edwin Li and William C. Wimley for valuable discussions. This work was supported by NIH grant GM068619, NSF grant MCB 0315663 and Research Scholar grant RSG-04-201-01 from the American Cancer Society (to K. H.).


  1. Adams PD, Arkin IT, Engelman DM, Brunger AT (1995) Computational searching and mutagenesis suggest a structure for the pentameric transmembrane domain of phospholamban. Nat Struct Biol 2(2):154–162PubMedCrossRefGoogle Scholar
  2. Blume-Jensen P, Hunter T (2001) Oncogenic kinase signalling. Nature 411:355–365PubMedCrossRefGoogle Scholar
  3. Cho JY, Guo CS, Torello M, Lunstrum GP, Iwata T, Deng CX, Horton WA (2004) Defective lysosomal targeting of activated fibroblast growth factor receptor 3 in achondroplasia. Proc Natl Acad Sci USA 101:609–614PubMedCrossRefGoogle Scholar
  4. Deng C, Wynshaw-Boris W, Zhou F, Kuo A, Leder P (1996) Fibroblast growth factor receptor 3 is a negative regulator of bone growth. Cell 84:911–921PubMedCrossRefGoogle Scholar
  5. Fantl WJ, Johnson DE, Williams LT (1993) Signaling by receptor tyrosine kinases. Annu Rev Biochem 62:453–481PubMedGoogle Scholar
  6. Fisher LE, Engelman DM, Sturgis JN (1999) Detergents modulate dimerization, but not helicity, of the glycophorin A transmembrane domain. J Mol Biol 293:639–651PubMedCrossRefGoogle Scholar
  7. Han X, Mihailescu M, Hristova K (2006) Neutron diffraction studies of fluid bilayers with transmembrane proteins: structural consequences of the achondroplasia mutation. Biophys J 91(10):3736–3747PubMedCrossRefGoogle Scholar
  8. Iwamoto T, You M, Li E, Spangler J, Tomich JM, Hristova K (2005) Synthesis and initial characterization of FGFR3 transmembrane domain: Consequences of sequence modifications. Biochim Biophys Acta 1668:240–247PubMedCrossRefGoogle Scholar
  9. Lakowicz JR (1999) Principles of Fluorescence Spectroscopy. Kluwer Academic/Plenum Publishers, New YorkGoogle Scholar
  10. Li E, Hristova K (2004) Imaging FRET measurements of transmembrane helix interactions in lipid bilayers on a solid support. Langmuir 20:9053–9060PubMedCrossRefGoogle Scholar
  11. Li E, Hristova K (2006) Role of receptor tyrosine kinase transmembrane domains in cell signaling and human pathologies. Biochemistry 45:6241–6251PubMedCrossRefGoogle Scholar
  12. Li E, You M, Hristova K (2005) SDS-PAGE and FRET suggest weak interactions between FGFR3 TM domains in the absence of extracellular domains and ligands. Biochemistry 44:352–360PubMedCrossRefGoogle Scholar
  13. Li E, You M, Hristova K (2006) FGFR3 dimer stabilization due to a single amino acid pathogenic mutation. J Mol Biol 356:600–612PubMedCrossRefGoogle Scholar
  14. Magde D, Wong R, Seybold PG (2002) Fluorescence quantum yields and their relation to lifetimes of rhodamine 6G and fluorescein in nine solvents: Improved absolute standards for quantum yields. Photochem Photobiol 75:327–334PubMedCrossRefGoogle Scholar
  15. Merzlyakov M, You M, Li E, Hristova K (2006) Transmembrane helix heterodimerization in lipids bilayers: Probing the energetics behind autosomal dominant growth disorders. J Mol Biol 358:1–7PubMedCrossRefGoogle Scholar
  16. Meyers GA, Orlow SJ, Munro IR, Przylepa KA, Jabs EW (1995) Fibroblast-growth-factor-receptor-3 (Fgfr3) transmembrane mutation in Crouzon syndrome with Acanthosis nigricans. Nat Genet 11:462–464PubMedCrossRefGoogle Scholar
  17. Otsuki T, Nakazawa N, Taniwaki M, Yamada O, Sakaguchi H, Wada H, Yawata Y, Ueki A (1998) Establishment of a new human myeloma cell line, KMS-18, having t(4;14)(p16.3;q32.3) derived from a case phenotypically transformed from Ig A-lambda to BJP-lambda, and associated with hyperammonemia. Int J Oncol 12:545–552PubMedGoogle Scholar
  18. Raicu V, Jansma DB, Miller RJD, Friesen JD (2005) Protein interaction quantified in vivo by spectrally resolved fluorescence resonance energy transfer. Biochem J 385:265–277PubMedCrossRefGoogle Scholar
  19. Ronchetti D, Greco A, Compasso S, Colombo G, Dell’Era P, Otsuki T, Lombardi L, Neri A (2001) Deregulated FGFR3 mutants in multiple myeloma cell lines with t(4;14): Comparative analysis of Y373C, K650E and the novel G384D mutations. Oncogene 20:3553–3562PubMedCrossRefGoogle Scholar
  20. Schlessinger J (2000) Cell signaling by receptor tyrosine kinases. Cell 103:211–225PubMedCrossRefGoogle Scholar
  21. Shiang R, Thompson LM, Zhu Y-Z, Church DM, Fielder TJ, Bocian M, Winokur ST, Wasmuth JJ (1994) Mutations in the transmembrane domain of FGFR3 cause the most common genetic form of dwarfism, achondroplasia. Cell 78:335–342PubMedCrossRefGoogle Scholar
  22. Vajo Z, Francomano CA, Wilkin DJ (2000) The molecular and genetic basis of fibroblast growth factor receptor 3 disorders: The achondroplasia family of skeletal dysplasias, Muenke craniosynostosis, and Crouzon syndrome with acanthosis nigricans. Endocr Rev 21:23–39PubMedCrossRefGoogle Scholar
  23. van der Geer P, Hunter T, Lindberg RA (1994) Receptor protein-tyrosine kinases and their signal transduction pathways. Annu Rev Cell Biol 10:251–337PubMedCrossRefGoogle Scholar
  24. van Rhijin B, van Tilborg A, Lurkin I, Bonaventure J, de Vries A, Thiery JP, van der Kwast TH, Zwarthoff E, Radvanyi F (2002) Novel fibroblast growth factor receptor 3 (FGFR3) mutations in bladder cancer previously identified in non-lethal skeletal disorders. Eur J Hum Genet 10:819–824CrossRefGoogle Scholar
  25. Webster MK, Donoghue DJ (1996) Constitutive activation of fibroblast growth factor receptor 3 by the transmembrane domain point mutation found in achondroplasia. EMBO J. 15:520–527PubMedGoogle Scholar
  26. You M, Li E, Hristova K (2006) The achondroplasia mutation does not alter the dimerization energetics of FGFR3 transmembrane domain. Biochemistry 45:5551–5556PubMedCrossRefGoogle Scholar
  27. You M, Li E, Wimley WC, Hristova K (2005) FRET in liposomes: Measurements of TM helix dimerization in the native bilayer environment. Anal Biochem 340:154–164PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Mikhail Merzlyakov
    • 1
  • Lirong Chen
    • 1
  • Kalina Hristova
    • 1
  1. 1.Department of Materials Science and EngineeringJohns Hopkins UniversityBaltimoreUSA

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