Molecular modeling based mutagenesis defines ligand binding and specificity determining regions of fibroblast growth factor receptors

Biochemistry. 1995 Aug 22;34(33):10325-33. doi: 10.1021/bi00033a002.

Abstract

The fibroblast growth factor receptor 2 (FGFR2) and the keratinocyte growth factor receptor (KGFR) have different ligand binding specificities despite differing only in the second half of their immunoglobulin-like (Ig-like) domain III. Three-dimensional model structures were generated for domain III on the basis of variable (V) Ig domains. The region that differs between the two receptors is predicted to include two loops: one connects beta-strands F-G and is analogous to the complementarity determining region 3 (CDR3) of immunoglobulins; the other connects beta-strands D-E. These regions were targeted for mutagenesis. Single mutations in the F-G loop were found to only slightly alter ligand binding, whereas a double mutant, KGFR Y345-->S,Q348-->I, acquired significant affinity for bFGF. Notably, the affinity of this double mutant KGFR for KGF and aFGF was essentially unaltered. A mutant FGFR2, in which the D-E beta-hairpin (T319TDKEI) is replaced with the KGFR D-E beta-hairpin (S319SNA), has 9-fold reduced affinity for bFGF. These results demonstrate that the F-G or CDR3 analogous loop in FGFRs plays a key role in determining ligand binding and specificity. In addition, however, the protein loop connecting beta-strands D and E may also be involved in ligand binding. Several point mutations in FGFR2, shown recently to give rise to multiple inherited skeletal defects, are localized according to our models to the F-G or D-E loops of domain III. Our results strongly suggest that these naturally occurring mutations specifically alter ligand binding by FGFR2.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Binding Sites
  • Cross-Linking Reagents
  • Fibroblast Growth Factor 1 / metabolism
  • Fibroblast Growth Factor 10
  • Fibroblast Growth Factor 2 / metabolism
  • Fibroblast Growth Factor 7
  • Fibroblast Growth Factors*
  • Growth Substances / metabolism
  • Macromolecular Substances
  • Models, Molecular*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Protein Structure, Secondary
  • Receptor, Fibroblast Growth Factor, Type 2
  • Receptors, Fibroblast Growth Factor / chemistry*
  • Receptors, Fibroblast Growth Factor / genetics*
  • Receptors, Growth Factor / chemistry
  • Receptors, Growth Factor / genetics
  • Sequence Alignment
  • Structure-Activity Relationship

Substances

  • Cross-Linking Reagents
  • Fibroblast Growth Factor 10
  • Growth Substances
  • Macromolecular Substances
  • Receptors, Fibroblast Growth Factor
  • Receptors, Growth Factor
  • Fibroblast Growth Factor 2
  • Fibroblast Growth Factor 1
  • Fibroblast Growth Factor 7
  • Fibroblast Growth Factors
  • Receptor, Fibroblast Growth Factor, Type 2
  • keratinocyte growth factor receptor