Figure 1. Specificity of Gpr124 for Wnt7a and Wnt7b activation of canonical Wnt signaling via Lrp5 and Fz4 in transfected STF cells.

(A) Ratio of luciferase activity induced in STF cells by Wnt+Gpr124+Lrp5 compared to Wnt+vector (pRK5)+Lrp5 plotted against luciferase activity induced by Wnt+Gpr124+Lrp5. The means are shown from triplicate determinations. See Figure S1A for an enlargement of the lower left part of the plot. Super Top Flash (STF) cells are a stable HEK/293 derivative that expresses firefly luciferase in response to canonical Wnt signaling (Xu et al., 2004).

(B) Relative luciferase activity in STF cells of Wnt7a+Lrp5+Fz or Wnt7b+Lrp5+Fz with vector (pRK5) vs. Gpr124.

(C) Relative luciferase activity in STF cells of Gpr124 vs. Gpr125 with Wnt7a or Wnt7b in the presence of Fz4 and Lrp5.

(D) Co-culture of 293 cells transfected with Wnt7a, with or without Gpr124, together with STF cells transfected with Fz4 and Lrp5, with or without Gpr124.

(E) Comparison of Wnt7a/Fz4/Gpr124 vs. Norrin/Fz4/Tspan12 signaling and their dependence on Lrp5 vs. Lrp6 determined by relative luciferase activity in STF cells.

(F) Titrations of Gpr124, Wnt7a, Fz4, and Lrp5 or Lrp6 plasmids in transfected STF cells. For each titration, the three other plasmids were held at a standard concentration [per three wells of a 96 well tray, these are: Gpr124, 50 ng; Fz4, 50 ng; Wnt7a, 10 ng; Lrp5, 5 ng].

(G) Comparison of Gpr124 vs. Tspan12 effects on Wnt7a/Fz4/Lrp5 signaling in STF cells.

(H) Comparison of Gpr124 vs. Tspan12 effects on Norrin/Fz4/Lrp5 signaling in STF cells. For this experiment, Norrin was delivered in conditioned medium as a Norrin-alkaline phosphatase (AP) fusion protein.

(I) Illustration of plasma membrane proteins that mediate Wnt7a or Wnt7b/Fz4/Lrp5/Gpr124 andNorrin/Fz4/Lrp5/Tspan12 signaling. See also Figure S1 and table S1.