Functional studies of membrane-bound and purified human Hedgehog receptor Patched expressed in yeast.
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.
Joubert
Olivier
O
Laboratoire 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.
Nehmé
Rony
R
Fleury
Damien
D
De Rivoyre
Matthieu
M
Bidet
Michel
M
Polidori
Ange
A
Ruat
Martial
M
Pucci
Bernard
B
Mollat
Patrick
P
Mus-Veteau
Isabelle
I
D002462:Cell Membrane / Q000378:metabolism*; D053823:Hedgehog Proteins / Q000096:biosynthesis / Q000502:physiology*; D006801:Humans; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; D010446:Peptide Fragments / Q000096:biosynthesis; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000302:isolation & purification; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D000072086:Smoothened Receptor; D020349:Surface Plasmon Resonance
D053823:Hedgehog Proteins; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; D010446:Peptide Fragments; C000606240:Ptch1 protein, mouse; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C491023:SMO protein, human; C465570:Shh protein, mouse; D000072086:Smoothened Receptor; C544324:sonic hedgehog protein (2-198), mouse