FGF21 Activates Preferentially Adipose FGFR1-KLB Figure 4. Dose-dependent differential binding of FGF21, FGF19 and FGF1 to KLB or FGFR4 alone. (A) Interaction of FGF21, FGF19 and FGF1 with KLB. Graded concentrations as indicated for FGF21 (open square), FGF19 (not shown) and FGF1 (filled circle) were added together with 2 ng/ml 125I-FGF19 to the cells expressing KLB alone, and the remaining specific bindings of 125I-FGF1 under these conditions were then determined. (B) Competitive binding of FGF21 and FGF1 with FGF19 to FGFR4 alone. Graded concentrations as indicated for FGF21 (open square), FGF19 (not shown) and FGF1 (filled circle) were added together with 2 ng/ml 125I-FGF19 to the cells expressing FGFR4 alone, and the remaining specific bindings of 125I-FGF1 under these conditions were then determined. doi:10.1371/journal.pone.0033870.g004 but low in the hypothalamus. Skeletal muscle expresses very low level of FGFR1 predominantly as compared to the WAT and hypothalamus. The rather weak response of hypothalamus to exogenous FGF21 stimulation for 20 minutes may be due to the blood-brain barrier and the low level of KLB expression (Figures 8 and 9). The levels of KLB and FGFR1 in the tissues from KLB2/ 2 mice and WAT from FGFR1 CN mice, respectively, are below the detection limit, the arbitrary cycle time of 34 in qPCR analyses. Since FGF21 is preferentially expressed in the liver, our results indicate not only the critical role of KLB and FGFR1 as the biological receptor complex in transducing the signal of FGF21 and FGF19, but also a major emerging endocrine metabolic axis from the liver to adipose tissues. Discussion As a single transmembrane protein, KLB plays an important role in regulating bile acid synthesis, as revealed by genetic ablation in normal condition [18,60]. KLB2/2 mice exhibited elevated biosynthesis and secretion of bile acids through upregulation of Cyp7a1 expression, a rate-limiting enzyme in the bile acid anabolic pathway. This phenotype resembles those of the FGFR4 and FGF15 (FGF19 in human) knockout mice [11,24]. Mice deficient in KL exhibited a variety of aging-like phenotypes [19], many of which phenocopy those observed in FGF232/2 mice [61]. These studies uncovered the biological connections among the conventional HS-FGFR, the KL family, and the FGF19 subfamily that function in convergence in signal transduction pathways for commitment of metabolic regulation [9,10,56,62] rather than grossly the growth control. Like canonic FGFs, the endocrine FGF19, FGF21 and FGF23 remain to signal through FGFRs but only in the presence of the KL family. The underlying mechanism for the KL family integration and the consequence resulted from is still unclear. In this study, we found that although FGF19 and FGF21 are unable to bind HS-FGFR alone, they are able to bind through KLB with high affinity. The binding of FGF19 and FGF21 to KLB and FGFR-KLB is not affected by the presence of canonic member FGF1; similarly, the binding of FGF1 to FGFR and FGFR-KLB is also completely resistant to the presence of FGF19 and FGF21. The presence of KLB confers even higher affinity for the FGF19 subfamily on the HS-FGFR complex than KLB alone. These data suggest a mode for the endocrine FGF-initiated FGFR signaling complex formation that is different from that initiated by canonic FGFs. We speculate that the binding sites on FGFR for FGF1 and FGF19/21 may be not all the same, or may require additional composite ones during the complex assemblage [16,63]. It is also possible that FGF1 could reject the KLB from the complex, or that KLB has no effect on FGF1-FGFR complex formation even when present. The latter possibility appears to be supported by our data PLoS ONE | www.plosone.org 6 March 2012 | Volume 7 | Issue 3 | e33870