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 g rowth factor receptor (KGFR) have different ligand binding specificiti es despite differing only in the second half of their immunoglobulin-l ike (Ig-like) domain III. Three-dimensional model structures were gene rated for domain III on the basis of variable (V) Ig domains. The regi on that differs between the two receptors is predicted to include two loops: one connects beta-strands F-G and is analogous to the complemen tarity determining region 3 (CDR3) of immunoglobulins; the other conne cts beta-strands D-E. These regions were targeted for mutagenesis. Sin gle mutations in the F-G loop were found to only slightly alter ligand binding, whereas a double mutant, KGFR Y-345 --> S,Q(348) --> I, acqu ired significant affinity for bFGF. Notably, the affinity of this doub le mutant KGFR for KGF and aFGF was essentially unaltered. A mutant FG FR2, in which the D-E beta-hairpin (T(319)TDKEI) is replaced with the KGFR D-E beta-hairpin (S(319)SNA), has 9-fold reduced affinity for bFG F. These results demonstrate that the F-G or CDR3 analogous loop in FG FRs plays a key role in determining ligand binding and specificity. In addition, however, the protein loop connecting beta-strands D and E m ay also be involved in ligand binding. Several point mutations in FGFR 2, shown recently to give rise to multiple inherited skeletal defects, are localized according to our models to the F-C or D-E loops of doma in III. Our results strongly suggest that these naturally occurring mu tations specifically alter ligand binding by FGFR2.