Detection of Antigen Interactions Ex Vivo by Proximity Ligation Assay: Endogenous Dopamine D2-Adenosine A2A Receptor Complexes in the Striatum

Abstract

The existence of G protein-coupled receptor (GPCR) dimers and/or oligomers has been demonstrated in heterologous systems using a variety of biochemical and biophysical assays. While these interactions are the subject of intense research because of their potential role in modulating signaling and altering pharmacology, evidence for the existence of receptor interactions in vivo is still elusive because of a lack of appropriate methods to detect them. Here, we adapted and optimized a proximity ligation assay (PLA) for the detection in brain slices of molecular proximity of two antigens located on either the same or two different GPCRs. Using this approach we were able to confirm the existence of dopamine D2 and adenosine A2A receptor complexes in the striatum of mice ex vivo.

G protein-coupled receptor-receptor interactions give integrative dynamics to intercellular communication

“…in vivo). In this respect, a first example was the demonstration of A 2A -D 2 heteroreceptor complexes in the striatum of mice (Trifilieff et al, 2011) and rats (Fernandez-Dueñas et al, 2015), where the alteration of native A 2A -D 2 oligomers was also observed in experimental parkinsonism. Further examples (see Borroto-Escuela et al, 2017, for a detailed review) include the galanin Gal 1 -Gal 2 heteroreceptor dimer in the midbrain, the FGF 1 -5-HT 1A in the hippocampus, and the 5-HT 1A -5-HT 2A isoreceptor complex in cortical regions.…”

“…Biological methods for identifying GPCR oligomers in cells and tissues or in recombinant mammalian expression systems presently include energy transfer-based methods [fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET); Fernandez-Dueñas et al, 2012], bimolecular luminescence or fluorescence complementation (Gandia et al, 2008), total internal reflection fluorescence microscopy (Hern et al, 2010), fluorescence correlation spectroscopy (Chen et al, 2003), analysis of colocalization in immunohistochemical preparations (Agnati et al, 2005a), coimmunoprecipitation , assays based on bivalent ligands (Yekkirala et al, 2013), and in situ proximity ligation assays (Trifilieff et al, 2011).…”

The Existence of FGFR1–5-HT1A Receptor Heterocomplexes in Midbrain 5-HT Neurons of the Rat: Relevance for Neuroplasticity

“…In situ proximity ligation assay (PLA) was performed as described previously (Söderberg et al, 2006;Borroto-Escuela et al, 2010Trifilieff et al, 2011). Freefloating sections and RN33B cell cultures were used with the following primary antibodies: rabbit monoclonal anti-5HT1A (VTG Biosciences) and mouse monoclonal anti-FGFR1 (Abcam).…”

G Protein–Coupled Receptor Heterodimerization in the Brain

“…In situ PLA has previously been performed to confirm the existence of striatal A 2A R-D 2 R heteromers (Trifilieff et al, 2011). The PLA technique involved the use of two primary antibodies of different species directed to either D 2 R or to A 2A R (Fig.…”

“…In situ PLA has been used to study proteins and protein-protein interactions in a range of applications (Leuchowius et al, 2010;Nilsson et al, 2010). In 2011, the method was employed to study GPCRs heteromers, mainly adenosine A 2A and dopamine D 2 receptor heteromers in striatal sections (Trifilieff et al, 2011) and dopamine D 2 R and D 4 R in transiently transfected HEK293T cells (Borroto-Escuela et al, 2011). In addition, the methods were employed to demonstrate for the first time the existence of FGFR1 and 5-HT1A receptor heterocomplexes in the rat hippocampus and dorsal and median raphe in the midbrain ( Fig.…”