Functional studies of membrane-bound and purified human Hedgehog receptor Patched expressed in yeast

• Published in: Biochimica et biophysica acta 2009-09, Vol.1788 (9), p.1813-1821
• Main Authors: Joubert, Olivier, Nehmé, Rony, Fleury, Damien, De Rivoyre, Matthieu, Bidet, Michel, Polidori, Ange, Ruat, Martial, Pucci, Bernard, Mollat, Patrick, Mus-Veteau, Isabelle
• Format: Article
• Language: English
• Subjects: Cell Membrane; Cell Membrane - metabolism; Fluorinated surfactants; Hedgehog Proteins; Hedgehog Proteins - biosynthesis; Hedgehog Proteins - physiology; Heterologous expression; Humans; Life Sciences; Membrane receptor; Neurons and Cognition; Patched; Patched Receptors; Patched-1 Receptor; Peptide Fragments; Peptide Fragments - biosynthesis; Protein Conformation; Receptors, Cell Surface; Receptors, Cell Surface - chemistry; Receptors, Cell Surface - isolation & purification; Receptors, G-Protein-Coupled; Receptors, G-Protein-Coupled - chemistry; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Smoothened Receptor; Sonic Hedgehog; Surface Plasmon Resonance
• ISSN: 0005-2736; 0006-3002; 0167-4889; 1879-2642
• DOI: 10.1016/j.bbamem.2009.05.009

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.