title: Visualizing G Protein-Coupled Receptor-Receptor Interactions in Brain Using Proximity Ligation In Situ Assay.
abstract: G protein-coupled receptors (GPCRs) constitute the largest family of plasma membrane receptors, thus representing the more investigated drug targets in the design of new therapeutic strategies. The existence of receptor-receptor interactions has revolutionized the field, since GPCR oligomerization might confer new intervention opportunities in pharmacotherapy. However, demonstrating the existence of such receptor-receptor interactions in native tissue has been a bottleneck in GPCR pharmacology. Here, we discuss an experimental approach, the proximity ligation in situ assay (P-LISA), which provides enough sensitivity to evaluate a receptor's close proximity within a named GPCR oligomer. Indeed, we provide a detailed step-by-step protocol for P-LISA experiments visualizing receptor-receptor interactions in brain slices. Additionally, we provide instructions for slide observation, data acquisition and quantification. Finally, we also discuss these critical aspects determining the success of the technique, namely the fixation process and the validation of the primary antibodies used. Overall, the P-LISA is a powerful and straightforward technique to visualize receptor-receptor interactions when performed under optimal conditions.
authors.affiliation: Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina, IDIBELL-Universitat de Barcelona, Barcelona, Spain.
authors.forename: Jaume
authors.initials: J
authors.lastname: Taura
authors.affiliation: Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina, IDIBELL-Universitat de Barcelona, Barcelona, Spain.
authors.forename: Víctor
authors.initials: V
authors.lastname: Fernández-Dueñas
authors.affiliation: Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina, IDIBELL-Universitat de Barcelona, Barcelona, Spain.|Department of Physiology, Faculty of Sciences, University of Ghent, Ghent, Belgium.
authors.forename: Francisco
authors.initials: F
authors.lastname: Ciruela
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D001921:Brain / Q000378:metabolism*; D007091:Image Processing, Computer-Assisted; D051379:Mice; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D016707:Tissue Fixation
chemical_list: D043562:Receptors, G-Protein-Coupled
keywords: GPCR; brain; oligomerization; proximity ligation assay