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Updated: Jun 22, 2026
Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists
Published on: November 15, 2013
Functional studies of membrane-bound and purified human Hedgehog receptor Patched expressed in yeast
Olivier Joubert1, Rony Nehmé, Damien Fleury
1Laboratoire de Biologie et de Physiopathologie des Systèmes Intégrés, CNRS FRE 3094, Faculté des Sciences, (Université de Nice - Sophia Antipolis), Parc Valrose, F-06108 Nice Cedex 2, France.
Abstract:
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.
Insights
Researchers expressed human Patched (hPtc) in yeast, achieving its native conformation for interaction with Sonic Hedgehog (Shh). This breakthrough enables large-scale hPtc production for studying Shh pathway dysfunction in diseases.
Area of Science:
- Molecular Biology
- Biochemistry
- Developmental Biology
Background:
- The Sonic Hedgehog (Shh) pathway is crucial for embryonic development and stem cell function.
- Dysregulation of Shh signalling, involving Patched (Ptc) and Smoothened (Smo) receptors, is linked to birth defects, cancer, and neurodegenerative diseases.
- Understanding Ptc's interaction with Smo is vital, but limited knowledge exists regarding Ptc's structure and function.
Purpose of the Study:
- To develop structure-function studies of the Shh pathway receptors, Ptc and Smo.
- To achieve heterologous expression and purification of human Ptc (hPtc) in a native conformation.
- To enable biochemical characterization of hPtc for therapeutic development.
Main Methods:
- Heterologous expression of human Ptc (hPtc) in Saccharomyces cerevisiae.
- Interaction studies of hPtc with purified Sonic Hedgehog (Shh) ligand in yeast membrane fractions.
- Surface Plasmon Resonance (SPR) technology to assess ligand-binding capabilities after purification using fluorinated surfactants.
Main Results:
- Successfully expressed functional human Ptc (hPtc) in yeast, maintaining its native conformation.
- Demonstrated that yeast-expressed hPtc interacts with its ligand, Shh.
- Confirmed that fluorinated surfactants preserve hPtc's ligand-binding ability post-purification.
Conclusions:
- This study reports the first successful heterologous expression and purification of a stable, native conformation of human Ptc.
- The developed method allows for the scale-up of hPtc production for comprehensive biochemical characterization.
- This work paves the way for developing novel therapeutics targeting Shh signalling pathway dysfunctions.
