Biology Reference
In-Depth Information
14.4
RESULTS AND DISCUSSION
It is now widely accepted that GPCRs can interact and form oligomers, and a grow-
ing body of evidence points to the functional importance of oligomeric complexes
for receptor trafficking, receptor activation, and G protein coupling in native tissues
(
Rivero-Muller et al., 2010
). The clinical significance of GPCR oligomerization has
also become more evident during recent years, leading to identification of receptor
oligomers as novel therapeutic targets (
Gonzalez-Maeso et al., 2008
). We have re-
cently established the spectral quantitative FRET analysis called lux-FRET method
to analyze receptor-receptor interaction with high spatial and temporal resolution in
live cells. From lux-FRET analysis, we can not only obtain the apparent FRET ef-
ficiencies but also receive information about the relative expression levels and can
correlate their oligomerization state with the effector response. Lux-FRET in com-
bination with microscopy provides unique possibility to monitor protein-protein in-
teraction in living cells at a higher resolution in real time under physiological
conditions. Based on quantitative lux-FRET, we have developed custom-tailored ac-
quisition protocols that are optimized according to biomedical application and are
important in quantitative molecular microscopy. Noteworthy, this approach allows
to calculate and visualize (
Fig. 14.8
;
Renner et al., 2012
) the apparent FRET efficien-
cies for fractions of donors and acceptors over a wide range of donor molar fraction.
Examples of microscopic FRET images measured at LSM 780, spinning disk, Wide-
field, and TIRF setting with above mentioned defined acquisition protocols are
shown in
Fig. 14.9
. This analysis strategy now enables us to apply this technique
further for various biologically interesting single-cell experiments.
5-HT
7
-CFP and 5-HT
1A
-YFP
5-HT
1A
-CFP and 5HT
7
-YFP
0.25
0.25
0.2
0.2
0.15
0.15
0.1
0.1
0.05
0.05
0
0
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
1
x
D
x
D
Ef
D
Ef
A
FIGURE 14.8
Dimerization of 5-HT
1A
and 5-HT
7
receptors investigated by lux-FRET. Apparent FRET
efficiencies Ef
D
(blue) and Ef
A
(green) were calculated and are shown as functions of the
donor mole fraction x
D
for 5-HT
1A
-5-HT
7
receptors heteromers. Experimental data were fitted
according model for dynamic oligomerization described in Ref.
Renner et al. (2012)
.
