Rapid, Direct SMOOTHENED Activity Assays in Live Cells Using cAMP-Based Conformational Sensors

Madison F Walker; Benjamin R Myers

doi:10.1007/978-1-0716-1701-4_15

Rapid, Direct SMOOTHENED Activity Assays in Live Cells Using cAMP-Based Conformational Sensors

Methods Mol Biol. 2022:2374:175-184. doi: 10.1007/978-1-0716-1701-4_15.

Authors

Madison F Walker¹, Benjamin R Myers²

Affiliations

¹ Departments of Oncological Sciences, Biochemistry, and Bioengineering, University of Utah School of Medicine, Salt Lake City, UT, USA.
² Departments of Oncological Sciences, Biochemistry, and Bioengineering, University of Utah School of Medicine, Salt Lake City, UT, USA. benjamin.myers@hci.utah.edu.

PMID: 34562252
DOI: 10.1007/978-1-0716-1701-4_15

Abstract

Communication between PATCHED1 (PTCH1) and SMOOTHENED (SMO) is fundamental to Hedgehog (Hh) signal transduction in development and disease. We describe a real-time cell-based SMO functional assay based on SMO activity-dependent changes in cellular cAMP concentrations. This assay is capable of detecting changes in SMO conformation within minutes of PTCH1 inactivation by Hh ligands. As a result, it expands the range of experimental perturbations that can be used to dissect PTCH1-SMO communication, enabling a deeper mechanistic understanding of a longstanding mystery in Hh signal transduction.

Keywords: G proteins; GloSensor; Luminescence; Real-time readout; cAMP.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Cyclic AMP
Hedgehog Proteins
Ligands
Receptors, G-Protein-Coupled
Signal Transduction
Smoothened Receptor / metabolism*

Substances

Hedgehog Proteins
Ligands
Receptors, G-Protein-Coupled
Smoothened Receptor
Cyclic AMP

Grants and funding

R35 GM133672/GM/NIGMS NIH HHS/United States