Abstract
Receptor function is dependent on interaction with various intracellular proteins that ensure the localization and signaling of the receptor. While a number of approaches have been optimized for the isolation, purification, and proteomic characterization of receptor–protein interaction networks (interactomes) in cells, the capture of receptor interactomes and their dynamic properties remains a challenge. In particular, the study of interactome components that bind to the receptor with low affinity or can rapidly dissociate from the macromolecular complex is difficult. Here we describe how chemical crosslinking (CC) can aid in the isolation and proteomic analysis of receptor–protein interactions. The addition of CC to standard affinity purification and mass spectrometry protocols boosts the power of protein capture within the proteomic assay and enables the identification of specific binding partners under various cellular and receptor states. The utility of CC in receptor interactome studies is highlighted for the nicotinic acetylcholine receptor as well as several other receptor types. A better understanding of receptors and their interactions with proteins spearheads molecular biology, informs an integral part of bench medicine which helps in drug development, drug action, and understanding the pathophysiology of disease.
Highlights
Reviewed by: Asha Suryanarayanan, Manchester College School of Pharmacy, USA Gopalkumar Rakesh, National Institute of Mental Health and Neurosciences, India
While a number of approaches have been optimized for the isolation, purification, and proteomic characterization of receptor–protein interaction networks in cells, the capture of receptor interactomes and their dynamic properties remains a challenge
The addition of chemical crosslinking (CC) to standard affinity purification and mass spectrometry protocols boosts the power of protein capture within the proteomic assay and enables the identification of specific binding partners under various cellular and receptor states
Summary
To biochemically study receptor–protein interactions, one must be able to isolate receptors and their interactomes from the lipid plasma membrane. This process is challenging because of the need to keep diverse protein–protein interactions intact during the extraction and purification of the protein complex. Large polypeptide membrane spanning receptors, such as ligand gated ion channels, demand strong detergent-based solubilization in order to ensure extraction of all receptor subunits. The stringency of these detergent conditions, can lead to a loss in numerous receptor–protein associations.
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