Biology Reference
In-Depth Information
The results of coimmunoprecipitation were individually visualized by Western blot
analysis with appropriate antibodies.
12.1.1.3
Coimmunoprecipitation results
We examined using coimmunoprecipitation whether adenosine A
1
and TP
recep-
tors form hetero-oligomers. Using cells cotransfected with myc-A
1
R and HA-TP
a
,
we detected myc-A
1
R in the complex precipitated with anti-myc antibodies, which
suggested the validity of the method used here (
Mizuno, Suzuki, Hirasawa, &
Nakahata, 2012
). Interestingly, HA-TP
a
was observed in the complex precipitated
with anti-myc antibodies only in the cells cotransfected with myc-A
1
R and HA-
TP
a
did not form an inappropriate aggregation, we ex-
plored the immunoprecipitation using the cells transfected with HA-TP
a
. To confirm that HP-TP
a
alone
and the cells cotransfected with myc-A
1
R and HA-LPA1R, which did not interact
with each other. In these cases, we did not detect a specific band in the complex pre-
cipitated with anti-myc antibodies, which indicated that HP-TP
a
a
and myc-A
1
R
formed a specific interaction.
12.1.2
BRET
2
assay
12.1.2.1
BRET
2
assay to analyze GPCR oligomerization
BRET
2
is a method to sensitively detect receptor oligomerization in intact living
cells. Biophysical assays based on RET are valuable to overcome the issues of arti-
ficial aggregation in the coimmunoprecipitation described earlier. A large number of
GPCRs have recently been reported to form homo- or hetero-oligomerization using
RET (
Pfleger & Eidne, 2006
).
The efficiency of RET depends on the degree of the spectral overlap and the dis-
tance between the donor and acceptor. BRET typically occurs in the 1-10 nm region,
which is consistent with the distance in which two molecules can interact with each
other (
Fig. 12.1
). BRET
2
is an advanced BRET technology that uses Rluc as the
Oligomerization
GPCR
Rluc
Rluc
GFP
2
GFP
2
515 nm
emission
395 nm
emission
395 nm
Substrate
Substrate
BRET
2
FIGURE 12.1
A schematic representation of the principle of BRET
2
in GPCR oligomerization. BRET
2
consists of nonradiative energy transfer between a donor, Renilla luciferase (Rluc), and
an acceptor, modified green fluorescent protein (GFP
2
). Excitation of the donor results in the
emission of light by the acceptor only if the two molecules are in close proximity (10100
˚
).
