Biology Reference
In-Depth Information
13.3
DISCUSSION
With CoIP and BRET experiments, we and others could show oligomerization of
both families of taste receptors, TAS1Rs and TAS2Rs (
Jiang et al., 2004, Kuhn
et al., 2010, Nelson et al., 2002
). The demonstration of receptor complexes could
therefore be made with two different and independent techniques; both methods have
their advantages and disadvantages.
CoIP is an often-used biochemical technique and can be used for analysis of
protein-protein interactions
in vitro
and
in vivo
. When differently tagged or marked
receptors are used
in vitro
, homo-oligomerization can be investigated and hetero-
oligomerization. However, the use of detergents to lyse cells and solubilize mem-
brane proteins could lead to artificial aggregation of proteins during the process;
one point of critique is that one cannot be sure that the protein complexes occurred
already in the cells under physiological conditions (
Jordan &Devi, 1999, Milligan &
Bouvier, 2005
). Another critical point is that one has to ensure that all membrane
proteins have been fully solubilized and that no intact membrane patches are still
present in the preparation as this might lead to false-positive results (
Milligan &
Bouvier, 2005
). To address these points, we used only a mild detergent, incorporated
an ultracentrifugation step into our protocol, and included a control with mixed ly-
sates of cells expressing only one receptor. As a result, this procedure is rather long
and requires a lot of hands-on time, which makes the protocol only suitable for a
small number of samples.
BRET on the other hand is a newer biophysical technique and is adaptable for
higher throughput, as shown in this protocol. The BRET method requires the gener-
ation of special receptor fusion proteins and can therefore only be employed for
in vitro
studies. Homo- and hetero-oligomerization can be investigated. One definite
advantage over CoIP experiments is that the analysis is performed in living cells and
under physiological conditions (
Pfleger & Eidne, 2005
). A point of critique is, how-
ever, that BRET experiments do not show, like CoIP analysis, for example, direct
protein-protein interactions but only demonstrate close proximity of the investigated
receptor proteins.
Both methods together can overcome most of the limitations of the single
methods and together provide compelling evidence for oligomerization, like here
for TAS1R and TAS2R taste receptors. It is therefore advised to use both methods
for oligomerization studies. If only one method can be used, it would be best to con-
sider using CoIPs for investigating only few samples; no special equipment is
needed. For many samples, however, it might be better to use BRET. BRET requires
a luminometer with dual-emission detection and special filter sets and a coelenter-
azine as substrate. But it has the advantages of being suitable for a higher throughput
and of analyzing proteins in cells under physiological conditions.
CoIPs could, for example, also be used for studies of taste receptor oligomeriza-
tion
in vivo
. Recently, TAS2R receptor-specific antibodies became commercially
available and have already been successfully used to stain receptor proteins in taste
tissue (
Behrens et al., 2012
). These antibodies could theoretically also be used for
