br Disp Cleavage and Membrane Trafficking Knowledge

Disp Cleavage and Membrane Trafficking Knowledge of how Disp is regulated in Shh-producing HJC 0350 synthesis to control ligand deployment has, until recently, remained limited. Early studies examining Disp function in Madin–Darby Canine Kidney (MDCK) cells and Drosophila tissue suggested that it is likely to assemble into trimers and localizes to the basolateral membrane of polarized cells to release ligand 32., 33.. Deletion mutagenesis of Disp revealed that both localization and trimerization were dependent on the intracellular carboxyl-terminal tail [32]. This suggests that the carboxyl-terminal domain could function as a control point for Disp activity. However, post-translational modifications occurring in the tail have yet to be reported, making potential regulatory behaviors of the domain difficult to predict. Based on its predicted structural homology with Ptch, which binds Shh through its large EC loops, it is possible that Disp may also engage ligand through its EC domains 22., 24., 25., 29.. The EC loops of Disp could therefore provide another potential control point for regulation of Hhs release. Consistent with this hypothesis, a conserved Furin cleavage site was recently identified in the predicted first EC loop of Disp (Figure 1B). Cleavage by Furin occurs on the cell surface to generate the ~150-kDa carboxyl-terminal functional domain and to release a ~35-kDa fragment containing the first TM domain and a portion of EC1 (Figure 2A). Mutation of the cleavage site, or genetic loss of Furin, disrupted Disp cleavage and compromised Hhs release, confirming a functional link between Disp processing and functionality [34]. The mechanism by which cleavage promotes Hhs release is not yet clear. One possibility is that cleavage governs Disp–Shh binding. However, wild-type and cleavage-deficient Disp proteins bound Shh with similar efficiencies, indicating that cleavage is not a prerequisite for ligand association [34]. The ability of Disp to form high-molecular-weight complexes was also unaffected by cleavage-site mutation, making Furin-mediated Disp processing unlikely to control self-association and/or potential binding partner engagement. A behavior of Disp that was observed to be altered by cleavage disruption was its membrane trafficking. Examination of the subcellular localization of wild-type and cleavage-compromised Disp proteins in vivo in Drosophila revealed a change from predominantly basal and basolateral membrane enrichment of the wild-type protein to a uniform distribution of the cleavage-site mutant throughout all cellular membrane domains [34]. Cleavage-compromised Disp showed reduced colocalization with the early endosomal marker Ras-related protein 5 (Rab5), suggesting that Furin-mediated cleavage may guide membrane trafficking by controlling Disp endocytosis and/or membrane retargeting [34]. This possibility is supported by in vivo studies in Drosophila salivary glands. Whereas coexpression of Hh-GFP with wild-type Disp in salivary gland cells depleted the GFP signal from the tissue, cleavage-deficient Disp coexpression led to ligand retention on salivary gland cell membranes [34]. Moreover, overexpression of wild-type Disp in Drosophila wing imaginal discs triggered anterior wing overgrowth, suggesting potentiation of the long-range signal. Overexpression of non-cleavable Disp did not induce wing overgrowth, suggestive of attenuated Hh release. Thus, Furin processing is crucial for Disp to effectively deploy Hh to elicit long-range effects in vivo [34]. Whether Disp is cleaved by additional proteases has not yet been reported. Biochemical and genetic studies suggest a role for protein sheddases in Hhs release, raising the possibility that Disp could undergo further processing by this class of proteases. However, in these studies Hh ligands were found to be the substrates for the sheddases. The sheddases clipped the ligands' membrane-embedded cholesterol and palmitate modifications to free Hhs from their lipid membrane tethers 35., 36., 37.. The role of Disp in this model remains unclear. It could function by situating Hhs in the membrane such that sheddases can access the ligands' lipid modifications. It is also possible that Disp may assist by recruiting Scube2, which is proposed to aid in ligand release by influencing Hhs interaction with sheddase proteins 35., 36.. Future investigations are needed to determine whether Disp is also clipped by sheddases and to reconcile the proposed release of unlipidated Hhs with the role of the amino-terminal palmitate during Ptch binding 22., 23., 24., 25..