Biology Reference
In-Depth Information
Concentrations of Plasmids for the Competitive BRET 2 Assay
Table 12.2
HA-A 1 R-
Rluc
HA-A 1 R-
GFP 2
Unfused Receptor
Plasmids
2
/
HA-A 1 R-Rluc/HA-A 1 R-GFP
1
1
0, 0.4, 0.8, 1.2, 1.6, 2.0
HA-A 1 R
2
/
HA-A 1 R-Rluc/HA-A 1 R-GFP
1
1
0, 0.4, 0.8, 1.2, 1.6, 2.0
HA-TPa
2
/
HA-A 1 R-Rluc/HA-A 1 R-GFP
1
1
0, 0.6, 1.2, 1.8, 2.7, 3.6
HA-LPA1R
10 5 cells)
Adjust the amount of total plasmids using vector plasmids.
Plasmids ( m g/5
5
universal microplate an-
alyzer (Perkin Elmer Life Sciences) for the detection of Rluc at 410 nm and GFP 2 at
515 nm. Receptor association calculated as the BRET ratio are emission at 515 nmover
emission at 410 nm. The values were collected by subtracting the background signal
detected using nontransfected cells in the following equation:
m
M. Assays were conducted immediately using a Fusion
a
ð
515nmemission
control 515nm emission
Þ
BRET 2 ratio
¼
ð
410nmemission
control 410nm emission
Þ
12.1.2.3.2 Results of the competitive BRET 2 assay
The expression of HA-TP
or HA-A 1 R in cells coexpressing Rluc- and GFP 2 -fused
HA-A 1 R decreased the BRET 2 signals for homo-oligomerization between HA-A 1 R-
Rluc and HA-A 1 R-GFP 2 ( Fig. 12.2 A and B). In contrast, the expression of HA-
LPA1R did not eliminate the BRET 2 signal for A 1 R receptor homodimerization
( Fig. 12.2 C). These results suggest that the adenosine A 1 receptor selectively formed
a homo-oligomer or hetero-oligomer with the TP
a
receptor. Thus, the formation
of hetero-oligomerization could be demonstrated in living cells.
a
12.2 MEASUREMENT OF THE SIGNALING PATHWAY
12.2.1 Cyclic AMP assay
The pertussis toxin (PTX)-sensitive family of G proteins, G i/o proteins, inhibits cy-
clic AMP (cAMP) production. The adenosine A 1 receptor is mainly coupled to G i/o
proteins ( Ralevic & Burnstock, 1998 ). To investigate whether the formation of
hetero-oligomerization between adenosine A 1 and TP
a
receptors affects signaling
by the adenosine A 1 receptor via G i/o , we examined intracellular cAMP levels using
cells coexpressing both receptors.
The protocol for the cAMP assay was as follows. HEK293T cells were seeded
onto 48-well plates at a density of 2
10 4 cells/well. The cells were transfected with
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