Molecular modeling based mutagenesis defines ligand binding and specificity determining regions of fibroblast growth factor receptors.
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.
Gray
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TE
Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
Eisenstein
M
M
Shimon
T
T
Givol
D
D
Yayon
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D016220:Fibroblast Growth Factor 1 / Q000378:metabolism; D051526:Fibroblast Growth Factor 10; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D051523:Fibroblast Growth Factor 7; D005346:Fibroblast Growth Factors; D006133:Growth Substances / Q000378:metabolism; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D051497:Receptor, Fibroblast Growth Factor, Type 2; D017468:Receptors, Fibroblast Growth Factor / Q000737:chemistry* / Q000235:genetics*; D017978:Receptors, Growth Factor / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D003432:Cross-Linking Reagents; D051526:Fibroblast Growth Factor 10; D006133:Growth Substances; D046911:Macromolecular Substances; D017468:Receptors, Fibroblast Growth Factor; D017978:Receptors, Growth Factor; D016222:Fibroblast Growth Factor 2; D016220:Fibroblast Growth Factor 1; D051523:Fibroblast Growth Factor 7; D005346:Fibroblast Growth Factors; D051497:Receptor, Fibroblast Growth Factor, Type 2; C065104:keratinocyte growth factor receptor