Capture of D2 Dopamine Receptor Signaling Complexes in Striatal Cells for Mass Spectrometry Proteomic Analysis

Abstract

In recent years advancements in proteomic techniques have contributed to the understanding of protein interaction networks (Interactomes) in various cell types. Today, high throughput proteomics promises to define virtually all of the components of a signaling and a regulatory network within cells for various molecules including membrane-spanning receptors. The D2 dopamine receptor (D2R) is a primary mediator of dopamine transmission in the brain. Signaling through D2Rs has been linked to dopamine-mediated effects on motivation, reward, locomotion and addiction to drugs of abuse. In the striatum, the D2R is a key mediatory of dopamine transmission. Actions on this receptor are an important pharmacological property of various drugs including typical antipsychotics and drugs of abuse. Here we provide an approach for the identification protein interaction networks of the D2R within striatal cells. We discuss key assays and techniques, such as cellular membrane protein fractionation, western blot analysis, magnetic bead coimmunoprecipitation, and liquid chromatography electrospray ionization (LC-ESI) mass spectrometry, that can be used for the isolation and characterization of D2R protein interaction networks. This approach presents a reliable method for the identification and characterization of D2R signaling within cells.