Evaluating the Activity of Smoothened Toward G Proteins Using [³⁵S]Guanosine 5'-(3-O-thio)triphosphate ([³⁵S]GTPγS)

Methods Mol Biol. 2015:1322:35-44. doi: 10.1007/978-1-4939-2772-2_4.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • GTP-Binding Proteins / chemistry
  • GTP-Binding Proteins / metabolism*
  • Guanosine 5'-O-(3-Thiotriphosphate)* / chemistry
  • HEK293 Cells
  • Humans
  • Immunoprecipitation / methods
  • Protein Binding
  • Receptors, G-Protein-Coupled / chemistry
  • Receptors, G-Protein-Coupled / metabolism*
  • Smoothened Receptor
  • Sulfur Radioisotopes / chemistry

Substances

  • Receptors, G-Protein-Coupled
  • SMO protein, human
  • Smoothened Receptor
  • Sulfur Radioisotopes
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • GTP-Binding Proteins