Activities of the Cytoplasmic Domains of Patched-1 Modulate but Are Not Essential for the Regulation of Canonical Hedgehog Signaling

Abstract

The Hedgehog (Hh) pathway is a highly conserved signaling cascade crucial for cell fate determination during embryogenesis. Response to the Hh ligands is mediated by the receptor Patched-1 (Ptch1), a 12-pass transmembrane glycoprotein. Despite its essential role in Hh signaling and its activity as a tumor suppressor, Ptch1 remains largely uncharacterized. We demonstrate here that Ptch1 binds to itself to form oligomeric structures. Oligomerization is mediated by two distinct, structurally disordered, intracellular domains spanning amino acids 584 -734 ("middle loop") and 1162-1432 (C terminus). However, oligomerization is not required for Ptch1-dependent regulation of the canonical Hh pathway operating through Smo. Expression of a mutant protein that deletes both regions represses the Hh pathway and responds to the addition of Hh ligand independent of its inability to bind other factors such as Smurf2. Additionally, deletion of the cytoplasmic middle loop domain generates a Ptch1 mutant that, despite binding to Hh ligand, constitutively suppresses Hh signaling and increases the length of primary cilia. Constitutive activity because of deletion of this region is reversed by further deletion of specific sequences in the cytoplasmic C-terminal domain. These data reveal an interaction between the cytoplasmic domains of Ptch1 and that these domains modulate Ptch1 activity but are not essential for regulation of the Hh pathway.

Structural basis for the recognition of Sonic Hedgehog by human Patched1

“…To obtain a sample suitable for structural study, we generated several constructs of human Ptch1 based on sequence conservation and functional characterizations (fig. S2) (8,46,47). Consistent with a previous observation in a cell-based binding assay that the C terminus-truncated mouse Ptch1 binds to ShhN with affinity similar to that of wild-type Ptch1 (Ptch1-WT) (8), the C terminal intracellular domain of mouse Ptch1 was shown to be dispensable for Ptch1-dependent regulation of canonical Hh signaling (46,47).…”

“…S2) (8,46,47). Consistent with a previous observation in a cell-based binding assay that the C terminus-truncated mouse Ptch1 binds to ShhN with affinity similar to that of wild-type Ptch1 (Ptch1-WT) (8), the C terminal intracellular domain of mouse Ptch1 was shown to be dispensable for Ptch1-dependent regulation of canonical Hh signaling (46,47). We therefore tested a number of C-terminus truncations and identified an optimal construct for human Ptch1 (residues 1 to 1305) that exhibited sufficient expression level and good solution behavior ( fig.…”

Autophagic Flux Is Regulated by Interaction Between the C-terminal Domain of PATCHED1 and ATG101

“…Both functional domains and the 12-TM “core” are necessary for inhibition of SMO in the absence of HH proteins. The PTCH1 CTD is dispensable for regulation of SMO (11); however, it is a critical regulatory domain responsible for the high turnover rate of PTCH1 (12, 13). The CTD binds to the ubiquitin E3 ligase Itch, which catalyzes ubiquitylation of K1426 to induce endocytosis and degradation of PTCH1 (13).…”

Inhibition of tetrameric Patched1 by Sonic Hedgehog through an asymmetric paradigm

“…Such architectural differences suggest that Ptch1 may not undergo the similar rotating mechanism as the bacterial RND transporters and the four Ptch1 protomers likely operate independently of each other. Supporting this notion, deletion of the intracellular domains led to monomerization of Ptch1, which nevertheless remained functional in cultured cell signaling assays 23 . ShhN binding can result in both Ptch1 inhibition and internalization from the primary cilia 14 .…”

“…Distinct from most bacterial RND transporters whose structures were resolved as trimers 22 , all the physiologically relevant Ptch1 structures were in monomeric form, despite Ptch1 was shown to be an oligomer in physiological condition 23 . The oligomerization of Ptch1 is mediated by the intracellular middle-loop domain (MLD) and C-terminal domain (CTD) 23 . It was reported that the CTD of Drosophila Ptc formed a trimer 24 .…”