Figure 2.

Proteolytic processing of N-terminal CW residues is a prerequisite for controlled Shh and Drosophila Hh release and biofunction. (A) Targeted deletion of the CW motif (Shh^Δ) diminishes protein release from the cell surface, but a protein with all five basic CW amino acids (blue) replaced by alanines (red, Shh^5xA) is strongly released even in the absence of Scube2. Shh^5xA + Scube2 was set to 100% and all other values are expressed relative to this value. One-way ANOVA; shown are average values ± SD, n = 4 for each data set. (B) UAS-CD8-GFP (green) produced in the anterior compartment or outside of the wing pouch under the control of GMR45433 and GMR45105, or in the posterior compartment under GMR48462 control. Cells in the posterior compartment produce endogenous Hh (red). Scale bar: 100 μm. (C) Adult Drosophila wings. In the normal wild-type situation (>CD8-GFP), the wing blade shows five longitudinal veins, an anterior cross vein, and a posterior cross vein. GMR-Gal4-induced Hh and Hh^3xA expression (note the presence of only three basic arginines in the fly CW-motif, labeled blue) causes anterior wing overgrowth or leads to overgrowth of the wing costa (arrows). In contrast, GMR-Gal4 overexpression of Hh^Δ did not cause such defects, confirming biological inactivity of the expressed protein. Scale bar: 1 mm.