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the accumulation of evidence that GPCRs form an oligomerization with a functional
alternationmay change the strategy for the discovery of novel drugs targetingGPCRs.
We previously targeted adenosine A
1
and thromboxane A
2
(TP) receptors to form a
functionally novel hetero-oligomer, since both receptors function in the same cells
(
Dare, Schulte, Karovic, Hammarberg, & Fredholm, 2007; Martinez-Salgado,
Garcia-Cenador, Fuentes-Calvo, Macias Nunez, & Lopez-Novoa, 2007; Nakahata,
2008
). Adenosine receptors have been subclassified intoA
1
,A
2A
,A
2B
,andA
3
subtypes
(
Fredholm, IJzerman, Jacobson, Klotz, & Linden, 2001
), and human thromboxane A
2
receptors have been divided into two subtypes, TXA
2
receptor
a
and TP
a
and TXA
2
receptor
(
Hirata et al., 1991
). This chapter describes themethods used to detect GPCR
oligomerization and alterations in the signaling pathways, principally according to our
findings on oligomerization between adenosine A
1
and TP
b
a
receptors.
12.1
DETECTION OF GPCR OLIGOMERIZATION
12.1.1
Coimmunoprecipitation
12.1.1.1
Coimmunoprecipitation of the GPCR oligomer
Coimmunoprecipitation has been the most frequently used and effective method for
studying GPCR oligomerization (
Kroeger, Pfleger, & Eidne, 2003
). This technique
requires the solubilization of cells expressing the two targeted GPCRs. Importantly,
the highly hydrophobic nature of the seven transmembrane domains makes the sol-
ubilization of GPCRs difficult. The detergent used must be strong enough to extract
GPCRs from cell membranes; however, it sometimes dissociates the oligomers.
There is also a risk of the detection of nonspecific complexes produced during this
step. It is important to carefully choose the detergent and examine the composition of
the solubilizing buffer. Additionally, the absence of coimmunoprecipitation should
be confirmed in cells expressing only one receptor of the target or coexpressing the
noninteracting partner (
Jordan & Devi, 1999
). We coimmunoprecipitated homo- and
hetero-GPCR oligomers using cells individually coexpressing target GPCRs with
distinct tags. In addition, we performed immunoprecipitation using lysates of the
cells expressing only one receptor of the target receptors to assess the validity of
the method used. We consequently obtained results to suggest that some GPCRs
form homo- and/or hetero-oligomers (
Suzuki, Namba, Mizuno, & Nakata, 2013;
Suzuki, Namba, Tsuga, & Nakata, 2006
).
In the following section, we described the protocol for the coimmunoprecipita-
tion of adenosine A
1
and TP
a
receptors.
12.1.1.2
Protocol for coimmunoprecipitation
Human embryonic kidney 293T (HEK293T) cells were seeded onto 10 cm culture
dishes at a density of 3.5
10
6
cells/dish. At 24 h post seeding, cells were transfected
with myc-A
1
R, HA-tagged TP
a
receptor (HA-TP
a
), and HA-LPA1R, respectively.
The transfected cells were collected by centrifugation at 1900
g
and washed twice
with Dulbecco's phosphate-buffered saline (
Table 12.1
). The cells were disrupted by
sonication using a Handy Sonic UR-20P (Tomy Seiko, Tokyo, Japan) in 300
m
lof
