title: Synthesis and initial characterization of FGFR3 transmembrane domain: consequences of sequence modifications.
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.
authors.affiliation: Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA.
authors.forename: Takeo
authors.initials: T
authors.lastname: Iwamoto
authors.forename: Min
authors.initials: M
authors.lastname: You
authors.forename: Edwin
authors.initials: E
authors.lastname: Li
authors.forename: Jamie
authors.initials: J
authors.lastname: Spangler
authors.forename: John M
authors.initials: JM
authors.lastname: Tomich
authors.forename: Kalina
authors.initials: K
authors.lastname: Hristova
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019281:Dimerization; D008239:Lysine / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000032:analysis* / Q000737:chemistry*; D051498:Receptor, Fibroblast Growth Factor, Type 3; D017468:Receptors, Fibroblast Growth Factor / Q000032:analysis* / Q000737:chemistry*; D013329:Structure-Activity Relationship
chemical_list: D008565:Membrane Proteins; D017468:Receptors, Fibroblast Growth Factor; D011505:Protein-Tyrosine Kinases; D051498:Receptor, Fibroblast Growth Factor, Type 3; D008239:Lysine