Biology Reference
In-Depth Information
7.2.5
Analysis of 5-HT
4
R dimers by TR-FRET
TR-FRET technology provides an easy way to detect the existence of 5-HT
4
dimers
and to examine their propensity to form heterodimers with other GPCRs.
Transfect COS-7 cells with the appropriate plasmids (N-terminally tagged
with HA or FLAG epitopes) and seed them in 96-well plates (100,000 cells/well).
Prepare 12 wells per condition. 24 h after transfection, wash the cells with HBS
and incubate them at 4
C for 24 h with the appropriate fluorescent anti-FLAG or
anti-HA antibodies diluted in HBS-KF (KF is added to avoid quenching of europium
cryptate). In half of the wells, add 50
m
L of 4 nM anti-FLAG M2-K and 50
m
Lof
10 nM anti-HA-d2. In the other half of the wells, add 50
m
L of 4 nM anti-FLAG
M2-K and 50
L of HBS-KF. Quantification of FRET signals is performed by
homogenous time-resolved fluorescence (HTRF
®
) settings (
Maurel et al., 2004
)
on appropriate apparatus (see
http://www.htrf.com/readers
for compatible readers).
Express the results as the specific signal over background, Delta F, as described in
Maurel et al. (2004)
.
Figure 7.4
presents the different type of experiments that can be routinely per-
formed using this simple and convenient technology. HA or FLAG-tagged GB
1
and GB
2
GABA
B
receptor subunits are classically used as positive controls of con-
stitutive dimerization. You have to verify that 5-HT
4
and GABA
B
receptors are
expressed at the cell surface in similar amounts by ELISA quantification for example
(
Fig. 7.4
A). In these conditions, the TR-FRET signal detected when we coexpress
HA- and FLAG-5-HT
4
R(
Fig. 7.4
B) represents only 30% of the signal obtained
for GABA
B
R heterodimers. Indeed, GABA
B
receptors expressed at the cell surface
are obligatory heterodimers, whereas HA-5-HT
4
R monomers could associate with
either HA-5-HT
4
or FLAG-5-HT
4
receptors. Therefore, HA-5-HT
4
R/FLAG-5-
HT
4
R dimers, which are the only couples producing FRET, represent only half of
the real amount of dimers at the cell surface. Thus, the real signal for 5-HT
4
R dimers
can be assumed to be around 60% of the GABA
B
R FRET signal.
By maintaining a constant density of HA-5-HT
4
Rs (donors, use anti-HA-K an-
tibodies) and increasing the density of FLAG-5-HT
4
R or FLAG-GB
2
(acceptors,
use anti-FLAG M2-d2), saturating FRET curves are obtained for WT 5-HT
4
recep-
tor, whereas the signal between 5-HT
4
R and GB
2
is linear and unsaturable
(
Fig. 7.4
C). These types of results indicate that 5-HT
4
R dimerization is specific,
whereas the low 5-HT
4
R/GB
2
signal reflects a collisional and nonspecific contact.
Competitions experiments can also be performed to assess specificity of 5-HT
4
R
dimerization. Coexpress constant amounts of HA- and FLAG-5-HT
4
R with increas-
ing amounts of competing GPCRs, N-terminally tagged with Myc epitope:
GABA
B2
R,
m
serotonin type 7 receptor
(5-HT
7
R), or
b
2
-adrenergic receptor
(
b
2
-AR), in
Fig. 7.4
D. As shown by its corresponding horizontal data-fitting line,
GABA
B2
R (GB
2
) is unable to compete with 5-HT
4
R dimerization (
Fig. 7.4
D).
5-HT
7
receptors compete with the formation of 5-HT
4
R homodimers at relatively
high and probably not physiological concentrations. Interestingly, Myc-
b
2
-AR
strongly reduces the FRET signal as efficiently as Myc-5-HT
4
R and competed with
