GPCR Oligomerization: Contribution to Receptor Biogenesis - GPCR Signalling Complexes: Synthesis, Assembly, Trafficking and Specificity - page 50

where GPCRs are interacting. Firstly, the intensity of luminescence is dim and
requires specialized equipment to measure. Therefore, current BRET technology
is not yet compatible with fluorescence microscopy, rendering visualization of
dimer biogenesis difficult. In addition, BRET is initiated by the addition of a
membrane-permeable substrate that allows the detection of the interaction in all
cellular compartments. As a consequence, BRET cannot provide information on
the subcellular site of interaction.

3.2.2.3 Bimolecular Fluorescence or Luminescence Complementation

(BiFC/BiLC)

Both biochemical and biophysical methods have been used to demonstrate the inter-
action between GPCRs; however, it is not clear whether the receptor is in a dimeric
or an oligomeric complex. Using a protein-fragment complementation approach,
receptor mosaics can be studied, which allow for the detection of three or more
interacting receptors. In a BiFC/BiLC assay, the donor and/or the acceptor protein
will be fragmented and genetically fused to the interacting proteins of interest.
When both fragments come together (e.g. upon GPCR dimerization) the protein
will be reconstituted to a functional FRET or BRET donor/acceptor and studying
compartmental localization will be possible (Ciruela et al. 2010 ). BiFC was able to
show that at least three adenosine A 2A receptors assemble into oligomers at the PM
in differentiated neuronal cells (Vidi et al. 2008 ) and at least four dopamine D 2
receptors are located in close molecular proximity at the PM of living mammalian
cells (Guo et al. 2008 ; Rajagopal et al. 2010 ) .

3.2.3 Proximity Ligation Assay (PLA)

Progress towards understanding GPCR trafficking in a physiological setting is fre-
quently complicated by the fact that these proteins are difficult to detect and manip-
ulate in their native environment. Better antibodies will allow the field to move from
heterologous overexpression systems to studies involving model organisms. For in
situ detection of heteromers, the Proximity Ligation Assay (PLA, Olink Bioscience)
can be applied using unconjugated primary antibodies from different species
(Fig. 3.3a ). The two secondary antibodies, each recognizing a specific species
epitope, are covalently coupled to oligonucleotides; the proximity MINUS and
PLUS probes. When both secondary antibodies are near each other, proximity-
dependent ligation and rolling circle amplification of a circular DNA reporter mol-
ecule occurs, which can then be visualized (Fig. 3.3b ). Using this approach, the
existence of a dopamine D 2 and adenosine A 2A receptor complex in the striatum of
mice was confirmed (Trifilieff et al. 2011 ). This method offers the opportunity to
perform a co-localization test with a cellular marker.