Stability study of the human G-protein coupled receptor, Smoothened.
Smoothened is a member of the G-protein coupled receptor (GPCR) family responsible for the transduction of the Hedgehog signal to the intracellular effectors of the Hedgehog signaling pathway. Aberrant regulation of this receptor is implicated in many cancers but also in neurodegenerative disorders. Despite the pharmacological relevance of this receptor, very little is known about its functional mechanism and its physiological ligand. In order to characterize this receptor for basic and pharmacological interests, we developed the expression of human Smoothened in the yeast Saccharomyces cerevisiae and Smoothened was then purified. Using Surface Plasmon Resonance technology, we showed that human Smoothened was in a native conformational state and able to interact with its antagonist, the cyclopamine, both at the yeast plasma membrane and after purification. Thermostability assays on purified human Smoothened showed that this GPCR is relatively stable in the classical detergent dodecyl-beta-d-maltoside (DDM). The fluorinated surfactant C(8)F(17)TAC, which has been proposed to be less aggressive towards membrane proteins than classical detergents, increased Smoothened thermostability in solution. Moreover, the replacement of a glycine by an arginine in the third intracellular loop of Smoothened coupled to the use of the fluorinated surfactant C(8)F(17)TAC during the mutant purification increased Smoothened thermostability even more. These data will be very useful for future crystallization assays and structural characterization of the human receptor Smoothened.
Nehmé
Rony
R
Université de Nice - Sophia Antipolis, CNRS UMR 6543, Parc Valrose, F-06108 Nice Cedex 2, France.
Joubert
Olivier
O
Bidet
Michel
M
Lacombe
Benoît
B
Polidori
Ange
A
Pucci
Bernard
B
Mus-Veteau
Isabelle
I
D019943:Amino Acid Substitution; D005960:Glucosides / Q000737:chemistry; D053823:Hedgehog Proteins / Q000737:chemistry / Q000235:genetics; D006801:Humans; D020125:Mutation, Missense; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism; D019636:Neurodegenerative Diseases / Q000235:genetics / Q000378:metabolism; D055550:Protein Stability; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D000072086:Smoothened Receptor; D020349:Surface Plasmon Resonance; D013501:Surface-Active Agents / Q000737:chemistry; D014704:Veratrum Alkaloids / Q000737:chemistry
D005960:Glucosides; D053823:Hedgehog Proteins; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; C491023:SMO protein, human; D000072086:Smoothened Receptor; D013501:Surface-Active Agents; D014704:Veratrum Alkaloids; C040358:dodecyl maltoside; C000541:cyclopamine