Dataset Information

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.

SUBMITTER: Lu X

PROVIDER: S-EPMC1578677 | biostudies-literature | 2006 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Lu Xingwu X Liu Songmei S Kornberg Thomas B TB

Genes & development 20060901 18

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 ...[more]

PMID: 16980583

Similar Datasets

Project description:BackgroundSonic hedgehog (Shh) signaling plays a crucial role in growth and patterning during embryonic development, and also in stem cell maintenance and tissue regeneration in adults. Aberrant Shh pathway activation is involved in the development of many tumors, and one of the most affected Shh signaling steps found in these tumors is the regulation of the signaling receptor Smoothened by the Shh receptor Patched. In the present work, we investigated Patched activity and the mechanism by which Patched inhibits Smoothened.Methodology/principal findingsUsing the well-known Shh-responding cell line of mouse fibroblasts NIH 3T3, we first observed that enhancement of the intracellular cholesterol concentration induces Smoothened enrichment in the plasma membrane, which is a crucial step for the signaling activation. We found that binding of Shh protein to its receptor Patched, which involves Patched internalization, increases the intracellular concentration of cholesterol and decreases the efflux of a fluorescent cholesterol derivative (BODIPY-cholesterol) from these cells. Treatment of fibroblasts with cyclopamine, an antagonist of Shh signaling, inhibits Patched expression and reduces BODIPY-cholesterol efflux, while treatment with the Shh pathway agonist SAG enhances Patched protein expression and BODIPY-cholesterol efflux. We also show that over-expression of human Patched in the yeast S. cerevisiae results in a significant boost of BODIPY-cholesterol efflux. Furthermore, we demonstrate that purified Patched binds to cholesterol, and that the interaction of Shh with Patched inhibits the binding of Patched to cholesterol.Conclusion/significanceOur results suggest that Patched may contribute to cholesterol efflux from cells, and to modulation of the intracellular cholesterol concentration. This activity is likely responsible for the inhibition of the enrichment of Smoothened in the plasma membrane, which is an important step in Shh pathway activation.

Project description:ObjectiveAstrocytes are glial cells proposed as the main Sonic hedgehog (Shh)-responsive cells in the adult brain. Their roles in mediating Shh functions are still poorly understood. In the hypothalamus, astrocytes support neuronal circuits implicated in the regulation of energy metabolism. In this study, we investigated the impact of genetic activation of Shh signaling on hypothalamic astrocytes and characterized its effects on energy metabolism.MethodsWe analyzed the distribution of gene transcripts of the Shh pathway (Ptc, Gli1, Gli2, and Gli3) in astrocytes using single molecule fluorescence in situ hybridization combined with immunohistofluorescence of Shh peptides by Western blotting in the adult mouse hypothalamus. Based on the metabolic phenotype, we characterized Glast-CreERT2-YFP-Ptc-/- (YFP-Ptc-/-) mice and their controls over time and under a high-fat diet (HFD) to investigate the potential effects of conditional astrocytic deletion of the Shh receptor Patched (Ptc) on metabolic efficiency, insulin sensitivity, and systemic glucose metabolism. Molecular and biochemical assays were used to analyze the alteration of key pathways modulating energy metabolism, insulin sensitivity, glucose uptake, and inflammation. Primary astrocyte cultures were used to evaluate a potential role of Shh signaling in astrocytic glucose uptake.ResultsShh peptides were the highest in the hypothalamic extracts of adult mice and a large population of hypothalamic astrocytes expressed Ptc and Gli1-3 mRNAs. Characterization of Shh signaling after conditional Ptc deletion in the YFP-Ptc-/- mice revealed heterogeneity in hypothalamic astrocyte populations. Interestingly, activation of Shh signaling in Glast+ astrocytes enhanced insulin responsiveness as evidenced by glucose and insulin tolerance tests. This effect was maintained over time and associated with lower blood insulin levels and also observed under a HFD. The YFP-Ptc-/- mice exhibited a lean phenotype with the absence of body weight gain and a marked reduction of white and brown adipose tissues accompanied by increased whole-body fatty acid oxidation. In contrast, food intake, locomotor activity, and body temperature were not altered. At the cellular level, Ptc deletion did not affect glucose uptake in primary astrocyte cultures. In the hypothalamus, activation of the astrocytic Shh pathway was associated with the upregulation of transcripts coding for the insulin receptor and liver kinase B1 (LKB1) after 4 weeks and the glucose transporter GLUT-4 after 32 weeks.ConclusionsHere, we define hypothalamic Shh action on astrocytes as a novel master regulator of energy metabolism. In the hypothalamus, astrocytic Shh signaling could be critically involved in preventing both aging- and obesity-related metabolic disorders.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets