Different Roles of N- and C-Termini in the Functional Activity of FGF21

Fibroblast growth factor 21 is a member of endocrine FGFs subfamily, along with FGF19 and FGF23. It is emerging as a novel regulator with beneficial effects on a variety of metabolic parameters, including glucose and lipid control. FGF21 activity depends on membrane protein beta Klotho that physically complexes with various FGF receptors, thus conferring them the ability to bind FGF21 and activate downstream signaling pathways. FGF21, like other FGFs, folds to a P-trefoil-like core region, with disordered N- and C-termini. In order to investigate their role in the activity of FGF21, we have constructed a series of deletion mutants and tested them for their ability to (1) bind beta Klotho, analyzed by surface plasmon resonance spectroscopy (2) signal through MAPK phosphorylation and inhibit apoptosis in 3T3-LI/beta Klotho fibroblasts (3) stimulate GLUTI mRNA upregulation and glucose uptake in 3T3-LI adipocytes. Binding studies with beta Klotho revealed that the interaction with the co-receptor involves the C-terminus, as progressive removal of amino acids from the carboxy end decreased affinity for beta Klotho. By contrast, removal of up to 17 amino acids from the N-terminus had no effect on the interaction with beta Klotho. Terminal deletions had greater effect on function, as deletions of six amino acids from the amino-terminus and only four from the carboxy-terminus each significantly impacted activity (10-fold). Of the extreme terminal truncations, with no detectable activity, AN 17 acted as competitive antagonist while Delta C20 did not. Our structure/function studies show that the C-terminus is important for beta Klotho interaction whereas the N-terminus likely interacts directly with FGF receptors.