Reprogramming FGFR isoform specificity in FGF2 by deep mutational scanning.

The fibroblast growth factor (FGF) family exhibits distinct yet overlapping specificities toward seven fibroblast growth factor receptor (FGFR) isoforms, forming highly complex signaling networks through broad binding and activation profiles. FGF2 plays important physiological roles and has considerable therapeutic potential; however, its pleiotropic and non-selective activation of FGFRs can lead to adverse effects. Therefore, molecular engineering of FGF ligands with controlled receptor isoform specificity is required. Here, we engineered FGF2 variants with altered FGFR isoform specificity using mRNA display-based deep mutational scanning (DMS), thereby enabling systematic evaluation of residues within the FGFR-binding interface that contribute to FGFR isoform specificity despite limited structural information. We demonstrate for the first time that FGFs with altered FGFR isoform specificity can be generated. Notably, FGFR isoform specificity proved malleable and could be reprogrammed by single-amino acid substitutions. These findings reveal that receptor selectivity in FGF2 is tunable and provide a framework for rationally designing FGF ligands with tailored FGFR isoform specificity, advancing the understanding and control of complex FGF/FGFR signaling networks.