Biology Reference
In-Depth Information
donor, the codon-humanized form of GFP (GFP
2
) as the acceptor, and bisdeoxycoe-
lenterazine (DeepBlueC) as the substrate instead of benzyl-coelenterazine, which has
often been used in the classic BRET technique (
Ayoub & Pfleger, 2010; Pfleger &
Eidne, 2006
). To apply BRET
2
to the study of GPCR oligomerization, Rluc and
GFP
2
must be fused to the GPCRs of interest, and the BRET
2
signal can be measured
in the cells expressing the target receptors.
12.1.2.2
BRET
2
saturation assay
The BRET saturation assay is performed using cells coexpressing a fixed amount of
the Rluc-fused receptor of interest and increasing amounts of another GFP-fused re-
ceptor (
Terrillon et al., 2003
). Oligomerization induces saturation of the BRET sig-
nal according to high concentrations of the GFP-fused receptor, whereas nonspecific
interactions cause a continuous rise in BRET signals with increasing amounts of the
Rluc-fused receptor in a pseudolinear pattern (
Mercier, Salahpour, Angers, Breit, &
Bouvier, 2002
). We previously examined GPCR oligomerization using the BRET
2
saturation assay with cells expressing the Rluc- or GFP
2
-fused adenosine A
1
recep-
tor. Consequently, it became possible to determine the specific BRET
2
signal for
homo-oligomerization of the adenosine A
1
receptor in living cells (
Suzuki et al.,
2009
), and this result supported the previous studies that showed indicating
homo-oligomerization of the adenosine A
1
receptor using other methods (
Ciruela
et al., 1995; Yoshioka, Hosoda, Kuroda, & Nakata, 2002
).
12.1.2.3
Competitive BRET
2
assay
Competitive BRET
2
can occur when BRET
2
between two partners expressed at a
fixed donor-acceptor ratio is inhibited by the increasing coexpression of nonfused
partners, while no inhibition was seen with a nonfused noninteracting protein
(
Marullo & Bouvier, 2007
). Therefore, the competitive BRET
2
assay allows distin-
guished oligomerization to be from random collisions between overexpressing
proteins.
We performed a competitive BRET
2
assay in HEK293T cells transiently trans-
fected with Rluc- and GFP
2
-fused HA-tagged adenosine A
1
receptor (HA-A
1
R)
using nonfused HA-TP
receptors
was assessed based on the competition of adenosine A
1
receptor homodimerization
with the heterodimerization of adenosine A
1
and TP
a
. The interaction between adenosine A
1
and TP
a
a
receptors in living cells.
12.1.2.3.1
Protocol for the competitive BRET
2
assay
HEK293T cells (5
10
5
cells/35 mm dish) were cotransfected with a fixed amount of
the HA-A
1
R-Rluc and HA-A
1
R-GFP
2
plasmids and increasing amounts of the unfused
plasmids, HA-A
1
R, HA-TP
a
,orHA-LPA1R(
Table 12.2
). Nontransfected cells were
used as a control. The cells were harvested and suspended in BRET
2
buffer at 48 h after
transfection (
Table 12.1
). Suspended cells were distributed in a white-walled 96-well
plate (OptiPlate, Perkin Elmer Life Sciences, Boston, MA) at a density of 1
10
6
cells/well and incubated for 20 min at 37
C. DeepBlueC (Perkin Elmer Life Sciences)
or coelenterazine 400A (Biotium Inc., CA) was then added at a final concentration of
