Biology Reference
In-Depth Information
method has several drawbacks. The solubilization of hydrophobic GPCRs can lead
to aggregation, which may be mistaken for oligomerization. Furthermore, the solu-
bilization process with detergents may dissociate between GPCRs (
Ramsay, Kellett,
McVey, Rees, & Milligan, 2002; Salim et al., 2002
).
The use of resonance energy transfer (RET) may improve the drawbacks associated
with coimmunoprecipitation. The RET technique allowed for the detection of sensitive
receptor-receptor interactions in living cells, real-time monitoring, and visualization
with microscopy (
Ayoub & Pfleger, 2010; Pfleger & Eidne, 2005
). Furthermore,
RET is valuable for observing receptor-receptor interactions and also ligand-receptor
interactions (
Albizu et al., 2010
). The initial approach for GPCR oligomerization
using RET has been the fluorescence RET (FRET) assay (
Overton & Blumer,
2000
). Receptor-receptor interactions can be monitored by measuring RET between
cyan fluorescent protein- and yellow fluorescent protein-fused receptors. Convention-
ally, RET-based techniques cannot clearly distinguish dimers from high-order oligo-
mers. However, three-chromophore FRET technique, which is one of the RET-based
techniques being developed, could extend to this issue (
Galperin, Verkhusha, &
Sorkin, 2004
). Additionally, bioluminescence RET (BRET) is a biophysical technique
that represents a powerful tool with which to measure protein-protein interactions
in living cells (
Albizu et al., 2010; Pfleger & Eidne, 2006
). BRET commonly resorts
to the use of
Renilla
luciferase (Rluc) as the donor. Rluc transfers energy to the green
fluorescent protein (GFP) as the acceptor in the presence of coelenterazine, the
substrate of Rluc. FRETmonitors the energy transfer between two fluorescent proteins,
while BRET can monitor without external excitation because it occurs after the
oxidation of the substrate. BRET can avoid some of the problems associated with
FRET, for example, photobleaching and the coincidence of the donor and acceptor
(
Pfleger & Eidne, 2006
). Therefore, GPCR oligomerization has been confirmed with
the BRET assay in a large number of studies (
Pfleger & Eidne, 2005
).
Recent studies on GPCR oligomerization have also demonstrated the paradigm of
structural studies. Using atomic force microscopy techniques, rhodopsin molecules,
which are categorized as class A GPCR, have been visualized as dimers in native
membranes. These findings also provided unequivocal evidence that receptor dimers
and multimers exist in native tissues (
Fotiadis et al., 2003
), although their functional
relevance to GPCR dimerization remains controversial (
Chabre, Deterre, &
Antonny, 2009; Chabre & le Maire, 2005; Gurevich & Gurevich, 2008
).
Thus, various assays are useful for the study of GPCR oligomerization, and we
should choose the best method to match the purpose. On the other side of GPCR olig-
omerization, it is important to translate
in vitro
observations to the physiologically
relevant functions of cells and tissues. However, this is still difficult because GPCRs
function in an intricate environment. Diversification of the signaling pathway, a re-
quirement for membrane trafficking and altering the ligand specificity, should be
taken into consideration to understand GPCR-mediated cell functions.
Clinical drugs in the market today have been produced based on the theory that
GPCR is coupled to a single G protein in 1:1 stoichiometry. GPCR oligomerization
can increase the diversity of cellular signaling and is likely to occur
in vivo
. Therefore,
