The C-terminal tail of the Hedgehog receptor Patched regulates both localization and turnover

Abstract

Patched (Ptc) is a membrane protein whose function in Hedgehog (Hh) signal transduction has been conserved among metazoans and whose malfunction has been implicated in human cancers. Genetic analysis has shown that Ptc negatively regulates Hh signal transduction, but its activity and structure are not known. We investigated the functional and structural properties of Drosophila Ptc and its C-terminal domain (CTD), 183 residues that are predicted to reside in the cytoplasm. Our results show that Ptc, as well as truncated Ptc deleted of its CTD, forms a stable trimer. This observation is consistent with the proposal that Ptc is structurally similar to trimeric transporters. The CTD itself trimerizes and is required for both Ptc internalization and turnover. Two mutant forms of the CTD, one that disrupts trimerization and the other that mutates the target sequence of the Nedd4 ubiquitin ligase, stabilize Ptc but do not prevent internalization and sequestration of Hh. Ptc deleted of its CTD is stable and localizes to the plasma membrane. These data show that degradation of Ptc is regulated at a step subsequent to endocytosis, although endocytosis is a likely prerequisite. We also show that the CTD of mouse Ptc regulates turnover.[Keywords: Patched; Hedgehog; Hedgehog receptor; protein multimerization; trimer; protein turnover] Supplemental material is available at http://www.genesdev.org.

Dose-dependent requirement of patched homologue 1 in mouse pancreatic beta cell mass

“…Previous studies have revealed that the carboxy-terminal cytoplasmic domain of Patched plays an essential role in blocking HH signalling in wing disc [13], as well as regulating Patched degradation and trafficking [14] in Drosophila. mes encodes a mutant PTCH1 protein lacking most of the carboxy-terminal 220 amino acids [13] and is considered a hypomorphic allele.…”

Essential basal cytonemes take up Hedgehog in the Drosophila wing imaginal disc

“…Among these regulated genes in the third instar wing disc is ptc, which is expressed in a band of approximately five to seven cells along the A/P border (Capdevila et al, 1994a;Mullor et al, 1997). Ptc is a Hh receptor (Lu et al, 2006;Marigo et al, 1996;Stone et al, 1996). dpp, another target, is expressed in a band of as many as five to six cells that is displaced from the border by approximately two to three cells (Raftery et al, 1991).…”

A Functional and Putative Physiological Role of Calcitriol in Patched1/Smoothened Interaction

“…However, studies showing that Shhinduced activation of Hh signaling results in internalization of Shh-bound Ptch (14,47) questions this prediction, because loss of membrane-associated Ptch should also result in loss of secretion of Smo-activating molecules. Therefore, it rather seems feasible that Ptch may secrete a negative regulator of Smo activity.…”

“…The SSD is essential for the function of Ptch (13). According to the oligomeric structures of SSD transporters Ptch proteins form stable trimers (14). Thus, it is tempting to speculate that Ptch is a multisubunit transporter whose activity indirectly regulates the function of Smo, e.g.…”