Analysis of Dispatched Protein Processing and Sonic Hedgehog Ligand Release

The 12-pass transmembrane protein Dispatched (DISP) is essential for Sonic Hedgehog (SHH) release from ligand-producing cells and is indispensable for establishment of the SHH morphogen gradient during tissue patterning. Regulatory events controlling DISP release of SHH are not yet fully characterized. We recently demonstrated that DISP is cleaved by FURIN proprotein convertase at a conserved site in its first extracellular loop. Mutation of the cleavage site attenuates DISP-mediated SHH release, which indicates that Furin cleavage is a positive step toward DISP protein maturation. In this chapter, we present a ligand release/retention protocol that allows for the analysis of DISP cleavage, DISP-mediated release of SHH ligand from producing cells, and secretion-dependent signal induction in target cells.