Characterization of binding sites for C3201 on FGFR1c and β-Klotho.

<p>(A) C3201 requires FGFR1c and β-Klotho to induce signaling as measured by ERK phosphorylation in L6 cells. L6 cells were selected for this characterization as they do not express any endogenous FGF receptors. Data are expressed as % ERK protein that is phosphorylated, as measured by MSD. (B) Characterization of C3201 binding site on β-Klotho. C3201 does not bind to murine β-Klotho. A series of human-mouse β-Klotho chimeras were co-transfected into L6 cells with human FGFR1c, and signaling was measured by ERK phosphorylation. The β-Klotho chimeras are represented by cartoons next to the ERK phosphorylation histograms, with murine sequence indicated as black and human sequence represented as white. The plasma membrane is indicated in grey. Data are represented as mean +/− SEM (n = 2). (C) Characterization of C3201 binding site in FGFR1c. C3201 does not signal through FGFR4. A series of FGFR1c/FGFR4 domain swap mutants were constructed and transiently transfected into L6 cells with human β-Klotho, and signaling was measured by ERK phosphorylation. The FGFR1c/FGFR4 domain swap mutants are represented by cartoons next to the ERK phosphorylation histograms, with FGFR1c sequences indicated as white and FGFR4 sequences represented as black. The plasma membrane is indicated in grey. Data are represented as mean +/− SEM (n = 2). (D) Predicted location of C3201 binding sites in the FGFR1c/β-Klotho complex. The predicted binding sites are indicated by cross-hatched areas on cartoon in FGFR1c domain D2 and the C-terminal region of β-Klotho domain KL2.</p>