Association of fibroblast growth factor receptor 1 with the adaptor protein Grb14 - Characterization of a new receptor binding partner

Using the cytoplasmic domain of fibroblast growth factor receptor 1 (FGFR1) as bait in a yeast two-hybrid screen, Grb14 was identified as a FGFR1 bind ing partner. A kinase-inactive mutant of FGFR1 failed to interact with Grb1 4, indicating that activation of FGFR1 is necessary for binding. Deletion o f the C-tail or mutation of both C-tail tyrosine residues of FGFR1 to pheny lalanine abolished binding and deletion of the juxtamembrane domain of the receptor reduced binding, suggesting that Grb14 binds to FGFR1 at multiple sites. Co. immunoprecipitation and in vitro binding assays demonstrated tha t binding of Grb14 to FGFR1 in mammalian cells was dependent on receptor ac tivation by fibroblast growth factor-2 (FGF-2), Deletion of the Src homolog y 2 (SH2) domain of Grb14 reduced but did not block binding to FGFR1 and el iminated dependence on receptor activation. The SH2 domain alone bound both FGFR1 and platelet-derived growth factor receptor, whereas full-length Grb 14 bound only FGFR1, suggesting that regions upstream of the SH2 domain con fer specificity for FGFR1. Grb14 was phosphorylated on serine and threonine residues in unstimulated cells, and treatment with FGF-2 enhanced this pho sphorylation. Expression of exogenous Grb14 inhibited FGF-2-induced cell pr oliferation, whereas a point-mutated form of Grb14 incapable of binding to FGFR1 enhanced FGF-2-induced mitogenesis, These data demonstrate an interac tion between activated FGFR1 and Grb14 and suggest a role for Grb14 in FGF signaling.