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Functional studies of membrane-bound and purified human Hedgehog receptor Patched expressed in yeast
Open accessAbstract
The Sonic Hedgehog (Shh) signalling pathway plays an important role both in embryonic development and in adult stem cell function. Inappropriate regulation of this pathway is often due to dysfunction between two membrane receptors Patched (Ptc) and Smoothened (Smo), which lead to birth defects, cancer or neurodegenerative diseases. However, little is known about Ptc, the receptor of the Shh protein, and the way Ptc regulates Smo, the receptor responsible for the transduction of the signal. To develop structure-function studies of these receptors, we expressed human Ptc (hPtc) in the yeast Saccharomyces cerevisiae. We demonstrated that hPtc expressed in a yeast membrane fraction is able to interact with its purified ligand Shh, indicating that hPtc is produced in yeast in its native conformational state. Using Surface Plasmon Resonance technology, we showed that fluorinated surfactants preserve the ability of hPtc to interact with its ligand after purification. This is the first report on the heterologous expression and the purification of a native and stable conformation of the human receptor Ptc. This work will allow the scale-up of hPtc production enabling its biochemical characterization, allowing the development of new therapeutic approaches against diseases induced by Shh signalling dysfunction.
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“(2) PTCH1 interacts with the cytosolic adaptor four and a half LIM domains 2 (FHL2; best known as DRAL), hence favoring the assembly of a CASP9-activating complex consisting of caspase recruitment domain family member 8 (CARD8; also known as TUCAN) and neural precursor cell expressed, developmentally down-regulated 4, E3 ubiquitin protein ligase (NEDD4) [352–354]”
From Molecular mechanisms of cell death: recommendations of the nomenclature committee on cell death 2018. (2018) · cites this paper as [352]
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Galluzzi, L., Vitale, I., Aaronson, S. A., Abrams, J. M., Adam, D., Agostinis, P., Alnemri, E. S., Altucci, L., Amelio, I., Andrews, D. W., et al. (2018) · Cell Death & Differentiation · doi:10.1038/s41418-017-0012-4 · Cited by 6,680
Metadata
- Authors
- Joubert, O., Nehmé, R., Fleury, D., De Rivoyre, M., Bidet, M., Polidori, A., Ruat, M., Pucci, B., Mollat, P., & Mus-Veteau, I.
- Affiliations
- Centre National de la Recherche Scientifique (FR)
- Galapagos (France) (FR)
- Université d'Avignon et des Pays de Vaucluse (FR)
- Laboratoire de Neurobiologie Cellulaire et Moléculaire (FR)
- Venue
- Biochimica et Biophysica Acta (BBA) - Biomembranes
- Publisher
- Elsevier BV
- Published
- 2009-09
- Volume
- 1788
- Issue
- 9
- Pages
- 1813-1821
- ISSN
- 0005-2736 (ISSN-L)
- OpenAlex
- W2066045166
- Cited by
- 17
- References
- 36
- Citations by year
- 2026: 1 · 2022: 1 · 2018: 1 · 2017: 1 · 2016: 2 · 2015: 3 · 2014: 1 · 2012: 1
- FWCI
- 0.94
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Biochimica et Biophysica Acta (BBA) - Biomembranes (publishedVersion)
- PubMed (publishedVersion)
- HAL (submittedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Hedgehog Signaling Pathway Studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Receptor Mechanisms and Signaling
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Wnt/β-catenin signaling in development and cancer
- Keywords
- Patched receptor, Sonic hedgehog signaling, Smoothened, heterologous expression in yeast, receptor purification, ligand binding, fluorinated surfactants, receptor conformation, structure-function analysis
- MeSH terms
- Patched Receptors, Patched-1 Receptor, Smoothened Receptor, Cell Membrane, Humans, Peptide Fragments, Protein Conformation, Receptors, Cell Surface, Saccharomyces cerevisiae, Surface Plasmon Resonance, Receptors, G-Protein-Coupled, Hedgehog Proteins
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