Differential Activation of Cysteine-Substitution Mutants of Fibroblast Growth Factor Receptor 3 Is Determined by Cysteine Localization

J Bone Miner Res. 2002 May;17(5):860-8. doi: 10.1359/jbmr.2002.17.5.860.

Abstract

Various human skeletal disorders are thought to be caused by mutations in fibroblast growth factor receptor 3 (FGFR3). These result in chronic FGFR3 hyperactivation and inhibition of bone growth. One such disorder, thanatophoric dysplasia, the most common form of sporadic, lethal dwarfism, is associated frequently with cysteine substitutions (G370C, S371C, and Y373C) in the extracellular juxtamembrane region of the receptor. These mutations have been suggested to induce disulfide-mediated receptor dimerization and constitutive activation. An adjacent cysteine substitution (G375C) leads to a less severe form of human dwarfism, achondroplasia, suggesting that the intensity of FGFR3 activation by these cross-links may be position dependent. To test this hypothesis, we have sequentially replaced each amino acid at positions 370-375 of FGFR3 with cysteine. Expression of each of these mutant forms in 293T cells led to their spontaneous, ligand-independent dimerization and increased basal phosphorylation. Wild-type (WT) FGFR3 became dimerized and phosphorylated only on FGF stimulation. Among the mutants, only two (G370C and S371C) caused high basal phosphorylation with significantly increased constitutive levels of mitogen-activated protein kinase (MAPK) phosphorylation and c-fos transcription. This activity was probably caused by mutant homodimer pairs, because WT-mutant heterodimers were observed only in the presence, but not in the absence, of FGF1. The high spontaneous activity of the mutants in positions 370-371, unlike those in 372-375, affirms their known involvement with thanatophoric dysplasia. We conclude that the G370C and S371C mutant receptors spontaneously dimerize in the correct spatial orientation required for effective signal transduction, whereas the 372-5 mutants, like the WT receptor, may achieve this orientation only on ligand binding.

MeSH terms

  • Achondroplasia / genetics
  • Achondroplasia / metabolism
  • Amino Acid Substitution
  • Animals
  • Base Sequence
  • Cell Line
  • Cysteine / chemistry*
  • Cysteine / genetics*
  • DNA, Complementary / genetics
  • Dimerization
  • Humans
  • Ligands
  • Phosphorylation
  • Point Mutation*
  • Protein Structure, Quaternary
  • Protein-Tyrosine Kinases*
  • Rats
  • Receptor, Fibroblast Growth Factor, Type 3
  • Receptors, Fibroblast Growth Factor / chemistry*
  • Receptors, Fibroblast Growth Factor / genetics*
  • Receptors, Fibroblast Growth Factor / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Signal Transduction
  • Thanatophoric Dysplasia / genetics
  • Thanatophoric Dysplasia / metabolism

Substances

  • DNA, Complementary
  • Ligands
  • Receptors, Fibroblast Growth Factor
  • Recombinant Proteins
  • FGFR3 protein, human
  • Protein-Tyrosine Kinases
  • Receptor, Fibroblast Growth Factor, Type 3
  • Cysteine