Synthesis and initial characterization of FGFR3 transmembrane domain: Consequences of sequence modifications

Takeo Iwamoto, Min You, Edwin Li, Jamie Spangler, John M. Tomich, Kalina Hristova

Research output: Contribution to journalArticle

Abstract

Receptor Tyrosine Kinases (RTKs) conduct biochemical signals via lateral dimerization in the plasma membrane, and defects in their dimerization lead to unregulated signaling and disease. RTK transmembrane (TM) domains are proposed to play an important role in the process, underscored by the finding that single amino acids mutations in the TM domains can induce pathological phenotypes. Therefore, many important questions pertaining to the mode of signal transduction and the mechanism of pathology induction could be answered by studying the chemical-physical basis behind RTK TM domain dimerization and the interactions of RTK TM domains with lipids in model bilayer systems. As a first step towards this goal, here we report the synthesis of the TM domain of fibroblast growth factor receptor 3 (FGFR3), an RTK that is crucial for skeletal development. We have used solid phase peptide synthesis to produce two peptides: one corresponding to the membrane embedded segment and the naturally occurring flanking residues at the N- and C-termini (TMwt), and a second one in which the flanking residues have been substituted with diLysines at the termini (TMKK). We have demonstrated that the hydrophobic FGFR3 TM domain can be synthesized for biophysical studies with high yield. The protocol presented in the paper can be applied to the synthesis of other RTK TM domains. As expected, the Lys flanks decrease the hydrophobicity of the TM domain, such that TMKK elutes much earlier than TMwt during reverse phase HPLC purification. The Lysines have no effect on peptide solubility in SDS and on peptide secondary structure, but they abolish peptide dimerization on SDS gels. These results suggest that caution should be exercised when modifying RTK TM domains to render them more manageable for biophysical studies.

Original languageEnglish (US)
Pages (from-to)240-247
Number of pages8
JournalBiochimica et Biophysica Acta - Biomembranes
Volume1668
Issue number2
DOIs
StatePublished - Mar 1 2005

Keywords

  • FGFR3
  • Receptor tyrosine kinase
  • Solid phase peptide synthesis
  • Terminal lysine

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

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