Analysis and Quantification of GPCR Heteroreceptor Complexes and Their Allosteric Receptor–Receptor Interactions Using Radioligand Binding Autoradiography

Abstract

G protein-coupled receptors (GPCRs) complexes and their allosteric receptor–receptor interactions represent a new fundamental principle in molecular medicine for integration of transmitter signals in the plasma membrane. The allosteric receptor–receptor interactions in heteroreceptor complexes give diversity, specificity, and bias to the receptor protomers due to conformational changes in discrete domains leading to changes in receptor protomer function and their pharmacology. Therefore, a novel understanding of the molecular basis of central nervous system diseases should consider this phenomena and new strategies for mental and neurodegenerative disorders treatment should target heteroreceptor complexes based on a new pharmacology with combined treatment, multi-targeted drugs and heterobivalent drugs. In this chapter, it is described a technique to visualize the majority of G protein-coupled receptor allosteric receptor–receptor interactions in sections of frozen brain tissue using receptor autoradiography. The basic procedure involves incubating slide-mounted tissue sections with radioligands, washing and drying of the sections with specifically bound ligands under conditions that preserve ligand binding, and visualizing and quantifying the binding sites in the tissues. Protocols for brain extraction and sectioning, radioligand exposure, autoradiogram generation, and data quantification are provided, as are the optimal incubation conditions for the autoradiographic visualization of allosteric receptor–receptor interactions using agonist and antagonist radioligands.