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. 2011 Aug;51(2):111-8.
doi: 10.2144/000113719.

Detection of antigen interactions ex vivo by proximity ligation assay: endogenous dopamine D2-adenosine A2A receptor complexes in the striatum

Pierre Trifilieff  1 , Marie-Laure Rives, Eneko Urizar, Rebecca A Piskorowski, Harshad D Vishwasrao, John Castrillon, Claudia Schmauss, Maria Slättman, Mats Gullberg, Jonathan A Javitch
Affiliations

Detection of antigen interactions ex vivo by proximity ligation assay: endogenous dopamine D2-adenosine A2A receptor complexes in the striatum

Pierre Trifilieff et al. Biotechniques. 2011 Aug.

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.

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Conflict of interest statement

Competing interests statement:

Maria Slättman and Mats Gullberg work for the company that developed the technique that has been adapted in this study.

Figures

Figure 1
Figure 1. The Duolink assay principle
(a) Two primary antibodies raised in different species are used to detect two target antigens of interest (here as examples, grey/green (left) against a target protein (green) and grey/yellow (right) against a second target protein (yellow)). (b) Each species-specific secondary antibody (dark grey and light grey, respectively) provided in the Duolink kit has a unique short DNA strand attached to it (black line). When the secondary antibodies are in close proximity, the DNA strands can interact through a subsequent addition of two other circle-forming DNA oligonucleotides (circular black line). The distance between the two secondary antibodies is a maximum of 16 nm as calculated from the number of nucleotides in the attached DNA arms. (c) After enzymatic ligation, the two added oligonucleotides are amplified via rolling circle amplification using a polymerase to yield a long concatemeric copy of the circle formed by ligation. (d) After the amplification reaction, labeled complementary oligonucleotide probes are added to highlight the product (red circles).
Figure 2
Figure 2. Detection of GPCRs ex vivo in striatal sections using PLA
D2R was detected in the dorsal striatum of WT mice (a and d). PLA signal from single confocal slice was virtually absent in D2R KO mice (b and e) but was strongly increased when the receptor was overexpressed in the striatum by viral gene transfer of a D2L-R-mVenus (AAV D2-mVenus) (c and f). Similarly A2AR detection in the striatum of WT mice gave a strong PLA signal (g and i) that was absent in A2AR KO mice (h and j). Note that the non-specific nuclear signal (see also SF 3) is similar among genotypes and is unrelated to the presence of primary or secondary antibodies. (k;l) Quantification of PLA signals for D2R (k; unpaired t-test: t= −5.7; p<0.01) and A2AR (l; unpaired t-test: t=−4.2; p<0.01) demonstrates the difference of PLA signal density between WT and KO mice. Scale bars=10 μm.
Figure 3
Figure 3. Detection of proximity ex vivo in striatal sections using PLA
Proximity detection was performed on striatal slices overexpressing the D2L-R-mVenus (AAV D2-mVenus) using antibodies directed against D2-R and GFP (a–c). This can lead to intraprotomer as well as potential interprotomer proximity. (a) PLA signal. (b) Direct fluorescence of mVenus. (c) merge. Proximity between D2R and A2AR was detected by PLA in the striatum of WT mice (d and g). The signal was virtually absent in both D2R (e and h) and A2AR (f and i) KO mice. (d–f are pseudocolors) (j) Quantification of PLA signals for D2R and A2AR proximity confirmed the significant difference of PLA signal density between WT and KO mice (ANOVA: p<0.01; post-hoc comparison: WT/A2A KO, p<0.01; WT/D2 KO, p=0.01; A2A KO/D2 KO, p=0.46). Scale bars=10 μm.
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