Biology Reference
In-Depth Information
5.
Incubate at 37
C for 4.5 h. We have tested many time points for the CRE-luc
assay and have found that 4.5 h is the earliest time point that hCG-dependent
LHCGR-mediatedCRE-luc activity can be robustly and consistently determined.
Thirty minutes prior to the end of incubation, prepare the steadylite plus solution
so it has reached room temperature by the end of the incubation period.
6.
Using a multistep pipette, add 100
l of steadylite plus solution to each well,
cover with foil, and place on a plate shaker for 10 min at
m
200 rpm with
variable rotation.
7.
Transfer the contents of the 96-well plate to a white 96-well plate and read
immediately using a plate-reading luminometer.
8.
To measure
pRL-CMV
reporter gene activity, add 50
m
l of coelenterazine
solution (10
l 0.5 M
EDTA) to each well, cover with foil, and place on a plate shaker for 10 min at
m
l1
m
g/
m
l coelenterazine, 5 ml 0.5 MHEPES pH 7.8, 400
m
200 rpm and variable rotation.
9.
Read using a plate-reading luminometer and save data.
10.
To analyze data, each CRE-luc activity is normalized to its internal pRL-CMV
value to control for any variations in transfection efficiency between wells. We
conduct all assays in quadruplicate with a minimum of three independent
experiments. Data is usually expressed as a percentage of maximum response or
as a fold change over basal response.
To demonstrate that a variance in the degree of functional rescue can be observed
when coexpressing receptors in either stable or transient fashion, we will describe a
comparison of cAMP-generated responses using three different modes of LHCGR
B
and LHCGR
S
coexpression. All data shown are normalized to the WT LHCGR
response generated using the same transfection conditions. When comparing signal
responses of cells transiently transfected with either WT LHCGR or LHCGR
B
with
LHCGR
S
, we observe a partial functional rescue by the transactivating mutant
LHCGR, approximating 57% of WT functional response (
Fig. 23.2
), consistent with
degrees of rescue observed with other LHCGR mutants that were also transiently
expressed (
Ji, Lee, Song, Conn, & Ji, 2002
). When employing stable cellular expres-
sion of one mutant receptor and transiently coexpressing the other, the degree of
functional rescue observed increases. In stable cells expressing LHCGR
S
tran-
siently transfected with LHCGR
B
, a further increase in the functional rescue is
observed, generating a cAMP response that is 63% of the WT response (
Fig. 23.2
).
However, when using cell lines that stably coexpress both LHCGR
B
and LHCGR
S
,
complete restoration of cAMP-dependent signaling is observed, with the transactivat-
ing mutant receptors signaling to 100% of cells stably expressing WT LHCGR
(
Fig. 23.2
). As stable coexpression of LHCGR
B
/LHCGR
S
results in a high level
of cells coexpressing receptors, it reveals that transactivation of LHCGR can facilitate
functional rescue of cAMP-mediated responses by mutant LHCGRs with restoration
to that of WT LHCGR. These results may explain why a recent study into LHCGR
did not observe the same degree of functional rescue when using the same mutant
LHCGRs (
Zhang et al., 2012
).
