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  • Structurally the termini of FGF are

    2021-12-04

    Structurally, the termini of FGF21 are critical for effective signaling. Progressive amino Metformin truncation at either end of the protein sequentially impairs FGF21 bioactivity, but via different mechanisms. Shortening at the N-terminus weakens its ability to activate the FGFR/KLB-complex but does not impede binding to KLB. In contrast, consecutive C-terminal shortening diminishes FGF21 interaction with KLB to eventually eliminate the receptor-complex engagement [15], [16]. The importance of these findings is underscored by the report that Fibroblast Activation Protein (FAP) degrades both termini to inactivate FGF21, and as such has been implicated in the pathophysiology of metabolic disease [17], [18], [19], [20]. The basis for receptor activation by FGF19 is generally believed to align with what has been established for FGF21. The N-terminal region of FGF19 influences FGFR specificity [21], [22] while its C-terminal part is involved in KLB binding [23]. The FGF21 variant lacking seventeen N-terminal amino acids (FGF2118−181, also known as ΔN17) is capable of antagonizing the actions of both hormones [9], [24], but the structural elements for KLB recognition by at least FGF21 are only recently being explored [25]. Curiously, the sequence identity in the C-terminal region of the two proteins is less than 40%, which is seemingly low for a common binding site. FGF19 and FGF21 effectively correct metabolic abnormalities in rodents, including regulation of glucose homeostasis [11], [13], [26], [27], [28]. In humans, FGF21 agonists improve dyslipidemia, insulin resistance, and body weight, while such studies for FGF19 agonists have yet to be communicated. Unexpectedly, no meaningful improvement of blood glucose was observed with FGF21 treatment in diabetic patients [29], [30], possibly due to insufficient dose intensity. Indeed, dose-proportional glucose lowering was observed in diabetic, non-human primates in an acute manner but only at supra-pharmacologic levels, beyond those clinically tested [31], [32]. It is thus plausible that FGF21 super-agonists might successfully reverse clinical hyperglycemia. In this regard, a protease-stabilized FGF21 analog improved blood glucose in obese, non-diabetic monkeys at doses where native FGF21 failed [33]. We sought to determine if there is a common basis to KLB binding for the C-terminal segments of FGF19 and FGF21, since these proteins are as different in amino acid sequence as they are alike. Such knowledge could be of great importance in achieving enhanced KLB affinity that might enable super-agonism. We began our investigation by determining the minimal fraction of FGF2118−181 necessary to fully antagonize FGF21 function. The recognition that the C-terminal 25 residues of FGF21 antagonized FGF19 and FGF21 activity substantially simplified the evaluation of the structural elements defining KLB binding in the full-length proteins, which are more than 180 amino acids in size. A full alanine scan (Ala-scan) of FGF19- and FGF21-based peptides identified specific amino acids essential to receptor activation, and the antagonism in cellular assays closely associated with binding affinity to the FGFR1/KLB complex in a cell-free system. The impact of specific alanine substitutions in antagonism correlated with changes in agonism when introduced to full-length FGF21. In vivo, the most potent FGF19-based peptide antagonist attenuated FGF19 and FGF21 signaling in adipose tissue and pancreas, and a FGF21-19A hybrid protein revealed improved metabolic efficacy in mice relative to native FGFs. Collectively, our results map the KLB binding elements in FGF19 and FGF21 at single amino acid resolution and specify a common functional signature by which these endocrine hormones signal via FGFR/KLB complexes, despite the appreciable difference in their native sequences.
    Materials and methods
    Results
    Discussion The initial reports associating members of the FGF superfamily with energy homeostasis first appeared for FGF19 [10] then FGF21 [13]. Each of these factors has reduced affinity for heparan-sulfate proteoglycans [44] to enable their endocrine mode of action [45], [46], [47], as opposed to the classical FGFs that function close to site of their synthesis [48]. Furthermore, the hormone-like FGFs require a tissue-specific transmembrane co-factor KLB [49]. KLB functions as an otherwise inert cell surface protein that anchors FGF19 and FGF21 to their target tissues to facilitate signaling via FGFRs. Still, the nanomolar potencies of FGF19 and FGF21 (Figure 3A,C and 3D) are paltry relative to the picomolar activities of FGF1 and FGF2 when studied in the same assays (Figs. S4A and S4B). Furthermore, and in contrast to FGF19 and FGF21, the activity of the latter proteins is largely heparin-dependent [50] and displays bell-shape dose responses (Fig. S4C). Another endocrine protein, FGF23, is also significantly more potent than FGF21 (Fig. S4B). Consequently, the emerging perspective is that conventional FGFs, and in particular FGF1 that also signals through FGFRs, can display potent endocrine biology [51], [52], [53]. Collectively, these observations indicate that much higher FGF19 and FGF21 metabolic potency might yet be achieved via optimization of their association with the FGFR/KLB complex.