Figure 2. Analyses of binding kinetic and affinity constant. T- Rex 293 cells expressing inducible FGFR1 and constitutive KLB (A) and hepatoma cells HR4 expressing FGFR4 and reduced level of KLB (B) were maintained in DMEM medium with 5% FBS. Binding kinetic analysis of radiolabeled FGF19 to the FGFR-KLB complexes on cell surfaces were done as described in the Materials and Methods. The binding affinity constant values were determined by the Scatchard plot. doi:10.1371/journal.pone.0033870.g002 whether FGF21 has the same ability to bind KLB as FGF19, graded concentrations of FGF21 were used to compete with 125I- FGF19 binding to KLB. FGF21 was able to displace 125I-FGF19 from KLB in a dose-dependent manner that is similar to the unlabeled FGF19; in contrast, FGF1 was unable to do so at all concentrations determined (Figure 4A). These results are consis- tent with the role of the C-terminus of FGF19 and FGF21 in the interaction with KLB [53,54,57], and suggest a mechanism that endocrine FGFs form complex with FGFRs through in part the binding to KLB with high affinity but not to HS motifs, while canonic FGFs need only HS motifs for the complex formation with FGFRs. Since our results shown that FGF19 could bind to FGFR4 alone with 10% efficiency but not FGFR1, we ask whether such a weak binding to FGFR4 is related to the mechanism of FGF1 binding. In fact, FGF1 displaced 125I-FGF19 from FGFR4 completely in a dose dependent manner similar to that of unlabeled FGF19, while FGF21 was unable to do so (Figure 4B). This is in contrast to the inability of both FGF19 and FGF21 to displace 125I-FGF1 from binding to FGFR4, FGFR4-KLB or FGFR1-KLB (Figure 3B). These data indicates that there may be a fundamental difference between the canonic FGF1 and endocrine FGF19 or FGF21 in the interaction with FGFRs, and such a weak FGF19 interaction may contribute to the receptor selectivity. As the interaction of both FGF19 and FGF21 with the binary FGFR-KLB complex is stronger than with KLB, and the N-terminus of FGF21 is important for interaction with FGFRs measured by the down- FGF21 Activates Preferentially Adipose FGFR1-KLB stream ERK1/2 activation but not without the presence of KLB [53,54], our results suggest that the integration of KLB is the key mechanism in the productive complex formation for the endocrine FGF19 and FGF21 with the canonic HS-FGFRs pre-complex residing across the plasma membrane. FGF21 activates downstream signaling of FGFR1-KLB but not FGFR4-KLB To determine whether the detectable surface receptor interac- tion leads to receptor downstream signal transduction, we assessed the downstream ERK1/2 activation in the same T-Rex 293 cells on which we performed the binding analyses. The ectopic expressions of FGFR1 and FGFR4 were induced by 10–30 ng/ ml Tet overnight to avoid extensive apoptotic effect in KLB co- expressing cells [4]. Consistent with the binding profile, FGF1 activates FGFR1-KLB, FGFR1, FGFR4-KLB and FGFR4 equally well but not KLB, and KLB has no inhibitory or stimulatory effects on FGF1 binding (Figure 1 and 3) and activity (Figure 5A). FGF19 activates both FGFR1-KLB and FGFR4-KLB efficiently and FGFR4 alone weakly but not FGFR1 alone as revealed by FGFR autophosphorylation and pERK1/2 (Figure 5A); while in marked contrast, although FGF21 bound to FGFR4-KLB very weakly, it could only activate the FGFR1- KLB expressing cells (Figure 5A), not the FGFR4-KLB, FGFR4 or FGFR1 expressing cells over the response level of the cells expressing KLB alone. Both FGF19 and FGF21 activate the KLB- expressing cells, albeit with a weaker potency, which is consistent with other reports [17,21] as the T-293 cells expressed the endogenous FGFR1 at a low level (unpublished observation) [17]. These results further support the idea that FGF21 is likely unable to activate FGFR4-KLB expressing cells. To investigate whether the effective receptor binding in transfected T-293 cells represents biological or physiological interactions, and therefore, activities, we determined the biological responses of cells expressing the endogenous FGFR and KLB to FGF19 and FGF21 stimulation with a comparable concentration used in the aforementioned binding assays. In differentiated 3T3- L1 adipocytes that co-express predominantly the FGFR1-KLB, FGF19, FGF21 and FGF1 activate the ERK1/2 to an equal extent, while in hepatoma cells co-expressing preferentially the FGFR4-KLB but not FGFR1-KLB, FGF21 is unable to activate the ERK1/2 response as compared to FGF19 and FGF1 (Figure 5B). This again indicates a potential difference in the endocrine activities of FGF19 and FGF21. In the in vivo situation, FGF19 may activate a broad type of tissues other than the liver, while FGF21 may restrict to the tissues related to adipose property or expressing predominantly the FGFR1-KLB. Furthermore, ERK1/2 activation levels in response to graded dosages of FGF19 and FGF21 in these cells indicated the half- maximum concentrations for FGF19 and FGF21 were 10 and 6 ng/ml, which is 0.5 and 0.3 nM, respectively, given that the molecular weight of both is about 20 kDa (Figure 6). These concentrations are comparable to the recombinant FGF21 and FGF19 used in other reports [53,54,56]. FGF21 activates predominantly the response of white adipose tissue To gain further insights into how FGF19 and FGF21, which differ in specific FGFR-KLB interaction, differentially impact in vivo endocrine metabolic effects, we determined their activities in regulating the c-Fos gene expression, which is a marker in early response to FGF19 stimulation [21], in different endocrine and metabolic tissues including the liver and white adipose tissue that PLoS ONE | www.plosone.org 4 March 2012 | Volume 7 | Issue 3 | e33870