Protocol for identifying physiologically relevant binding proteins of G-protein-coupled receptors

Bill X Huang; Hee-Yong Kim

doi:10.1016/j.xpro.2023.102691

Protocol for identifying physiologically relevant binding proteins of G-protein-coupled receptors

STAR Protoc. 2023 Dec 15;4(4):102691. doi: 10.1016/j.xpro.2023.102691. Epub 2023 Nov 4.

Authors

Bill X Huang¹, Hee-Yong Kim²

Affiliations

¹ Laboratory of Molecular Signaling, National Institute on Alcohol Abuse and Alcoholism, NIH, 5625 Fishers Lane, Rockville, MD 20852, USA. Electronic address: bhuang@mail.nih.gov.
² Laboratory of Molecular Signaling, National Institute on Alcohol Abuse and Alcoholism, NIH, 5625 Fishers Lane, Rockville, MD 20852, USA. Electronic address: hykim@nih.gov.

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.

Keywords: Mass Spectrometry; Protein expression and purification; Proteomics.

Published by Elsevier Inc.

MeSH terms

Carrier Proteins*
Cell Membrane
Immunoprecipitation
Mass Spectrometry
Receptors, G-Protein-Coupled* / genetics

Substances

Carrier Proteins
Receptors, G-Protein-Coupled