Patched 1 reduces the accessibility of cholesterol in the outer leaflet of membranes
- Stanford University School of Medicine, United States
- Oxford University, United Kingdom
- Nanyang Technological University, Singapore
- University of Texas Southwestern Medical Center, United States
Abstract
A long-standing mystery in vertebrate Hedgehog signaling is how Patched 1 (PTCH1), the receptor for Hedgehog ligands, inhibits the activity of Smoothened, the protein that transmits the signal across the membrane. We previously proposed (Kinnebrew et al., 2019) that PTCH1 inhibits Smoothened by depleting accessible cholesterol from the ciliary membrane. To directly test the effect of PTCH1 on accessible cholesterol, we measured the transport activity of PTCH1 using an imaging-based assay to follow the kinetics of cholesterol extraction from the plasma membrane of live cells by methyl-β-cyclodextrin. PTCH1 depletes accessible cholesterol in the outer leaflet of the membrane in a manner regulated by its ligand Sonic Hedgehog and the transmembrane potassium gradient. We propose that PTCH1 moves cholesterol from the outer to the inner leaflet of the membrane in exchange for potassium ion export. Our results show that proteins can change accessible cholesterol levels in membranes to regulate signaling reaction.
Data availability
No dataset was generated or used during this study (such as deep sequencing data, mass spectrometry data, structural coordinates or maps, genetic data or clinical trial data) that required deposition in a repository such as GenBank, the PDB, mass spec data repositories, or clinical data repositories. We have provided original, uncropped scans of immunoblots shown in Figures 2B, 4B, and Figure 3-figure supplement 1 in the Source Data Files. All other data generated are included in this study, with replicates and statistics described in the figure legends and methods.
Article and author information
Author details
Arun Radhakrishnan
Funding
Cancer Research UK (C20724)
- Christian Siebold
Ministry of Education, Singapore (MOE-T2EP30120-0002)
- Yasunori Saheki
National Science Foundation (Predoctoral Fellowship)
- Maia Kinnebrew
Ford Foundation (Predoctoral Fellowship)
- Giovanni Luchetti
Cancer Research UK (A26752)
- Christian Siebold
European Research Council (647278)
- Christian Siebold
National Institutes of Health (GM118082)
- Rajat Rohatgi
National Institutes of Health (GM106078)
- Rajat Rohatgi
National Institutes of Health (HL20948)
- Arun Radhakrishnan
Welch Foundation (I-1793)
- Arun Radhakrishnan
Leducq Foundation (19CVD04)
- Arun Radhakrishnan
Ministry of Education, Singapore (MOE2017-T2-2-001)
- Yasunori Saheki
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Duojia Pan, UT Southwestern Medical Center and HHMI, United States
Publication history
- Received: May 27, 2021
- Accepted: October 25, 2021
- Accepted Manuscript published: October 26, 2021 (version 1)
- Version of Record published: December 8, 2021 (version 2)
Copyright
© 2021, Kinnebrew et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Patched 1 reduces the accessibility of cholesterol in the outer leaflet of membranes
eLife 10:e70504.
https://doi.org/10.7554/eLife.70504
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