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Updated: Jul 11, 2025

Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding
Published on: June 9, 2017
Protocol for identifying physiologically relevant binding proteins of G-protein-coupled receptors
1Laboratory of Molecular Signaling, National Institute on Alcohol Abuse and Alcoholism, NIH, 5625 Fishers Lane, Rockville, MD 20852, USA.
Abstract:
G-protein-coupled receptors (GPCRs) are important therapeutic targets expressed on the cell surface. Here, we present a protocol for identifying physiologically relevant binding proteins of adhesion GPCR GPR110. We describe steps for in-cell chemical crosslinking, immunoprecipitation, and quantitative high-resolution mass spectrometry. Notably, we detail a label-free quantitation strategy that eliminates irrelevant interacting proteins using an inactive GPR110 mutant with impaired surface expression. Furthermore, we outline procedures for validating the identified partners. For complete details on the use and execution of this protocol, please refer to Huang et al. (2023).1.
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