CRISPR screens reveal lipid modulators of the G protein-coupled receptor smoothened
Abstract/Contents
- Abstract
- The Hedgehog (Hh) signaling pathway is a GPCR-associated signaling pathway critical for vertebrate development and adult tissue homeostasis. Numerous questions remain about how the activity and localization of Smoothened (Smo)—a core Hh pathway component and GPCR—is controlled and modulated. The advent of CRISPR represents an opportunity to use high-throughput approaches to uncover novel modulators of Smo and Hh signaling as a whole. Here I present the results of a CRISPR screen of lipid-related genes that uncovered several lipid metabolic pathways that influence Hh signaling activity through separate specific effects on Smo. I present evidence that sphingomyelin synthesis and cholesterol synthesis both impact Hh signaling by modulating Smo activity, a discovery that also provides insight into a central question remaining about the mechanisms of vertebrate Hh signaling: how can cholesterol, an abundant lipid in the plasma membrane, act as the endogenous ligand for Smo as has been hypothesized? Using toxin-based fluorescent sensors of membrane lipids, we established that ciliary sphingomyelin limits cholesterol accessibility in that organelle. We postulate that this small pool of accessible cholesterol—rather than the total membrane cholesterol—is responsible for activating Smo and driving Hh signaling downstream of Patched1. I also validate another prospective negative regulator of Hh signaling suggested by our screen: the N-glycan synthesis pathway. Protein N-glycosylation can have great functional importance, but the impact of N-glycosylation remains poorly understood for many proteins. I demonstrate that inhibition of this general pathway has a specific effect on Hh pathway activity and on Smo in particular, potentiating signaling by increasing Smo accumulation at the ciliary membrane in response to Hh ligand. Intriguingly, the impact of N-glycan synthesis inhibition on Smo is not a result of hypoglycosylation of Smo itself, but rather the result of loss of glycosylation on an alternative protein. Our CRISPR screen thus sheds light on the distinct impacts of three lipid-related pathways on Smo behavior and Hh signaling, suggesting that our screening methodology may be useful to interrogate the relationship between lipid metabolic networks and GPCR signaling more generally.
Description
| Type of resource | text |
|---|---|
| Form | electronic resource; remote; computer; online resource |
| Extent | 1 online resource. |
| Place | California |
| Place | [Stanford, California] |
| Publisher | [Stanford University] |
| Copyright date | 2021; ©2021 |
| Publication date | 2021; 2021 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Iverson, Ellen Jean |
|---|---|
| Degree supervisor | Rohatgi, Rajat |
| Thesis advisor | Rohatgi, Rajat |
| Thesis advisor | Harbury, Pehr |
| Thesis advisor | Pfeffer, Suzanne |
| Degree committee member | Harbury, Pehr |
| Degree committee member | Pfeffer, Suzanne |
| Associated with | Stanford University, Department of Biochemistry |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Ellen Iverson. |
|---|---|
| Note | Submitted to the Department of Biochemistry. |
| Thesis | Thesis Ph.D. Stanford University 2021. |
| Location | https://purl.stanford.edu/wm505vn4423 |
Access conditions
- Copyright
- © 2021 by Ellen Jean Iverson
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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