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 [35S]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.
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Acknowledgments
Our work on the use of G proteins by Smoothened is supported by the National Institutes of Health grant GM080396.
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Manning, D.R., Shen, F., Riobo, N.A. (2015). Evaluating the Activity of Smoothened Toward G Proteins Using [35S]Guanosine 5′-(3-O-thio)triphosphate ([35S]GTPγS). In: Riobo, N. (eds) Hedgehog Signaling Protocols. Methods in Molecular Biology, vol 1322. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2772-2_4
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DOI: https://doi.org/10.1007/978-1-4939-2772-2_4
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2771-5
Online ISBN: 978-1-4939-2772-2
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