Biology Reference
In-Depth Information
150
100
50
0
Tst LHCGR
B
-
/
Tst LHCGR
S
-
Tst LHCGR
B
-
/
Stb LHCGR
S
-
Stb LHCGR
B
-
/
Stb LHCGR
s
-
FIGURE 23.2
Functional rescue of LHCGR mutants by transactivation in vitro. The degree of functional
rescue by transactivation of LHCGR
B
and LHCGR
S
, as determined by CRE-luc activity,
correlates with the method of receptor expression. Cells expressing receptors either stably
(Stb) or transiently (Tst) as indicated, treated with hCG (10 nM) for 4.5 h. Each data
point represents the mean
SEM of three independent experiments, carried out
in quadruplicate. Each data point was normalized to % of maximum WT response.
23.2.3
Studying transactivation in vivo
Despite the extensive data demonstrating that transactivation functionally rescues
GPCR function at the cellular level, the critical question outstanding was if GPCR
homodimerization, via transactivation, is a physiologically relevant mode of receptor
function
in vivo
. To address this question, we employed transgenic technology to cre-
ate mouse models expressing these mutants and utilizing the previously generated
LhCGR
knockout (LuRKO) mouse model (
Zhang, Poutanen, Wilbertz, &
Huhtaniemi, 2001
), which produces a clear reproductive phenotype of delayed pu-
berty, hypogonadism, high LH, undetectable levels of testosterone, arrested sperm
development, and infertility. These phenotypic parameters provided an ideal back-
ground in which to assess whether transactivation of LHCGR by the targeted coex-
pression of LHCGR
B
and LHCGR
S
could rescue the hypogonadism and infertility
of the LHCGR-null male mice, thus providing definitive evidence that receptor di-
merization through transactivation is a physiologically required mechanism of
LHCGR activation and signaling.
23.2.3.1
Generation of BAC constructs
The first step of investigating receptor transactivation
in vivo
was to identify which
method of transgenesis to employ. We wanted to ensure that the integrity of the spa-
tial/temporal expression pattern of LHCGR
B
/LHCGR
S
was matched to that of
