Biology Reference
In-Depth Information
of deleting TM6/7 as this has been previously described to be a “hotspot” for inacti-
vating mutations of LHCGR (
Huhtaniemi & Themmen, 2000
) and is essential for G
protein-dependent coupling (
Sangkuhl, Schulz, Schultz, & Sch¨neberg, 2002
). This
resulted in the desired mutant capable of ligand binding but deficient of ligand-induced
signaling. As this LHCGR
S
contained a large TM deletion, we were concerned on the
impact this would have on trafficking to the cell surface. Hence, to aid plasma mem-
brane targeting, we inserted into its 5
0
-end the bovine prolactin leading sequence, as
this has been previously shown to aid trafficking of impaired receptors to the cell sur-
face (
Osuga, Kudo, Kaipia, Kobilka, & Hsueh, 1997
). As antibodies to most GPCRs
have issues with affinity and specificity, we introduced differential N-terminal tags to
the receptors for monitoring of receptor expression, with LHCGR
B
HA tagged and
LHCGR
S
FLAG tagged. For all experiments described detailing LHCGR transacti-
vation, we have conducted parallel experiments with an HA-tagged WT LHCGR as a
model of cis- and transactivation (
Fig. 23.1
).
An important point to reemphasize is that before assessing transactivation, it is
essential to ensure that the phenotypic defect of the individual mutant receptors used
in transactivation assays is as expected. Our approach has been to conduct ligand-
binding assays and second messenger functional assays coupled to rigorous assess-
ment of cell surface expression of the individually expressed LHCGR
B
and
LHCGR
S
. Once established, experiments to investigate receptor transactivation
can proceed.
23.2.1
Protein-protein interactions
Receptor transactivation requires the physical interaction of receptors; therefore,
when beginning to investigate the possibility of receptor transactivation, it is prudent
to assess whether the receptors in question can associate. As this chapter will focus
more on the assessment of the functional aspects of receptor transactivation, detailed
methodology for determining protein-protein interactions will not be discussed.
However, we and others have shown the LHCGR
B
and LHCGR
S
to associate
using coimmunoprecipitation (
Rivero-M¨ ller et al., 2010
) and bioluminescence res-
onance energy transfer (BRET), respectively (
Zhang, Guan, & Segaloff, 2012
).
23.2.2
Functional measurement of receptor transactivation in vitro
To assess the functional rescue by transactivation
in vitro
, we have tested various
methods for coexpressing the LHCGR
B
and LHCGR
S
in human embryonic kid-
ney (HEK293) cells. In our experience, the degree of functional rescue observed cor-
relates to the level of receptor coexpression. For that purpose, we will briefly
describe how we obtained stable cell lines to carry out our studies into LHCGR
transactivation.
To obtain stable cell lines to assess the functional rescue through transactivation
of LHCGR
B
and LHCGR
S
, we have used Lipofectamine
®
2000 transfection re-
agent and a standard Geneticin selection protocol. After plating HEK293 cells onto a
