FGF21 Activates Preferentially Adipose FGFR1-KLB Figure 1. Receptor specificity of FGF1, FGF19 and FGF21. (A) Comparative binding properties of FGF19 and FGF1. Parental T-Rex 293 cells and cells expressing FGFR1-KLB, FGFR1, FGFR4-KLB, FGFR4 and KLB upon 30 ng/ml Tet induction for FGFRs were incubated with 2 ng/ml 125I-labeled FGF1 (white rectangle) or 125I-FGF19 (black rectangle) as indicated. Cell surface bound radioactivity was determined by c–counter. Data are shown as mean 6 s.d. of three independent experiments. (B) Covalent Affinity chemical cross-linking was performed as described [4]. Cross-linking bands were indicated by bidirectional arrows. (C) Receptor binding properties of FGF21 revealed by competition binding with 125I-FGF19. FGF21, FGF19 and FGF1 at 1 mg/ml were incubated in the presence of 2 ng/ml 125I-FGF19 with parental cells and cells expressing FGFR1-KLB, FGFR1, FGFR4-KLB, FGFR4 and KLB. Specific cell surface binding and statistic analyses were determined as above. doi:10.1371/journal.pone.0033870.g001 KLB, which include cells co-expressing FGFR4-KLB and cells expressing KLB alone [4]. It should be noted that the activity of recombinant FGF19 and FGF21 is comparable to those currently used in other groups (personal communication). Radio-iodinated FGF19 exhibited an affinity with a Kd value of 303 pM to the FGFR1-KLB complex expressed on the T-Rex 293 cells [4] (Fig. 2A), and 778 pM to a hepatocellular carcinoma cells expressing FGFR4 and KLB [4,8] (Fig. 2B). The binding affinity constants of FGF19 are 3–7 times less than those of FGF1 [55], but are better than those from other experiments [53,54,56]. Since a same iodination procedure inactivated both mouse and human FGF21, we employed mutual comparative competition binding assay and iodinated FGF19 to address their receptor binding ability and specificity; however, it would be desirable in the next step to overcome this iodination problem, so the native binding constant can be deduced and compared to FGF19 in the direct binding assay on cells expressing endogenous FGFR and KLB. Dose-dependent competition of FGF21 with FGF19 for binding to FGFR1-KLB but comparably not to FGFR4-KLB To determine quantitatively the interaction nature of FGF19 and FGF21 with FGFR1 and FGFR4, we assessed the dose-dependent competition of FGF21 with FGF19 for receptor binding. 125I- FGF19 was co-incubated with graded concentrations of FGF21 on cells co-expressing KLB and inducible FGFR1 or FGFR4, respectively [4]. The remaining binding of FGF19 was measured quantitatively by radio-activity. FGF21 couldn’t compete with 125I- FGF19 for FGFR4-KLB interaction, with a half-maximum effective concentration that is out of valid calculation range. In marked contrast, FGF21 competes for binding to FGFR1-KLB with a half- maximum concentration of about 150 ng/ml, which is comparable to 110 ng/ml for unlabeled FGF19 for binding to both FGFR1- KLB and FGFR4-KLB (Figure 3A). On the other hand, FGF1 at all concentrations tested couldn’t compete with 125I-FGF19 for binding to FGFR1-KLB and FGFR4- KLB (Figure 3A). Conversely, both FGF21 and FGF19 couldn’t compete with 125I-FGF1 for binding to either FGFR1-KLB (Figure 3B) or FGFR4-KLB (Figure 3B inset) at all concentrations tested. This strongly indicates that the biochemical mechanisms underlying the receptor interaction are likely different between the heparin-binding canonic FGFs and KLB-binding endocrine FGFs. Interaction of FGF19 and FGF21 with KLB One notable feature of FGF19 is its high-affinity binding to KLB alone but not FGFR alone [4] (Figure 1A). To further assess PLoS ONE | www.plosone.org 3 March 2012 | Volume 7 | Issue 3 | e33870