Isolation and Detection of G Protein-Coupled Receptor (GPCR) Heteroreceptor Complexes in Rat Brain Synaptosomal Preparation Using a Combined Brain Subcellular Fractionation/Co-immunoprecipitation (Co-IP) Procedures
The isolation and characterization of GPCR heteroreceptor complexes, specially those present at the central nervous system, are of crucial relevance for the understanding of the molecular mechanisms behind several mental and neurodegenerative disorders. The existence of homo- and heteroreceptor complexes with allosteric receptor-receptor interactions increases the diversity of receptor function including recognition, trafficking, and signaling. This phenomenon increases our understanding of how brain function is altered through molecular integration of receptor signals. An alteration in specific heteroreceptor complexes or their neuronal localization is considered to have a role in the pathogenic mechanisms that lead to mental and neurological diseases, including drug addiction, depression, Parkinson’s disease, and schizophrenia. Therefore, it is fundamental to understand the appropriate localization and synaptic clustering of these GPCR heteroreceptor complexes. This chapter represents a workflow for the analysis of GPCR heteroreceptor complexes by means of combined use of differential centrifugation/co-immunoprecipitation in rat brain tissue. The combination of differential centrifugation/co-immunoprecipitation allows the separation and detection of GPCR heteroreceptor complexes present at synaptic sites from those found in intracellular compartments and vesicular pools. It is a reproducible protocol and produces reliable quantitative data.
Key wordsG protein-coupled receptors (GPCRs) GPCR heteroreceptor complexes Receptor-receptor interactions Heterodimerization Co-immunoprecipitation Subcellular Synaptic membrane protein Ultracentrifugation
This work has been supported by the Karolinska Institutets Forskningsstiftelser 2014/2015 to DOBE, by the Swedish Medical Research Council (62X-00715-50-3) and AFA Försäkring (130328) to KF and DOBE. DOBE belongs to Academia de Biólogos Cubanos.
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