title: Evaluating the Activity of Smoothened Toward G Proteins Using [³⁵S]Guanosine 5'-(3-O-thio)triphosphate ([³⁵S]GTPγS).
abstract: The utilization of heterotrimeric G protein, and in particular those of the Gi, family, by Hedgehogs through Smoothened has become increasingly clear. We describe here a method for evaluating the activity of Smoothened toward G proteins in membranes derived from human embryonic kidney-293 (HEK293) cells. The assay relies on receptor-promoted exchange of GDP for [(35)S]GTPγS on the Gα subunit. The assay is best suited for analysis of the constitutive activity of Smoothened, inverse agonism superimposed on this activity, and neutral antagonism superimposed on inverse agonism. The assay would also be suitable for several other applications requiring a proximal, quantifiable readout of Smoothened activity.
authors.affiliation: Department of Pharmacology, Perelman School of Medicine, University of Pennsylvania, 3620 Hamilton Walk, Pennsylvania, PA, 19104-6084, USA, manning@mail.med.upenn.edu.
authors.forename: David R
authors.initials: DR
authors.lastname: Manning
authors.forename: Feng
authors.initials: F
authors.lastname: Shen
authors.forename: Natalia A
authors.initials: NA
authors.lastname: Riobo
mesh_terms: D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000378:metabolism*; D000072086:Smoothened Receptor; D013462:Sulfur Radioisotopes / Q000737:chemistry
chemical_list: D043562:Receptors, G-Protein-Coupled; C491023:SMO protein, human; D000072086:Smoothened Receptor; D013462:Sulfur Radioisotopes; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins