Biology Reference
In-Depth Information
2.
Lipofectamine
®
2000 (Invitrogen, UK)
3.
Anti-HA.11 antibody (used at 1:300 dilution, raised in mouse, Covance c/o
Cambridge Bioscience, UK), anti-FLAG antibody (used at 1:500 dilution, raised
in rabbit, Sigma-Aldrich, UK), and anti-rabbit Alexa 488 and anti-mouse Alexa
647 secondary antibodies (1:1000 dilution, Invitrogen, UK)
4.
Dulbecco's Modified Eagle Media (D5976) supplemented with 10% fetal bovine
serum with or without 100 IU penicillin/0.1 mg streptomycin for maintenance of
cells and transfections, respectively (all Sigma-Aldrich, UK), and OptiMEM
(Invitrogen, UK)
5.
Geneticin (Invitrogen, UK) for cell line selection
6.
Recombinant human chorionic gonadotropin (hCG) (e.g., National Peptides and
Hormones Program, USA)
7. CRE-luciferase (CRE-luc)
reporter gene construct in
pcDNA3.1
and
pRL-CMV
reporter gene transfection efficiency control (Promega, UK)
8.
Steadylite plus reagent (Perkin-Elmer, UK) and coelenterazine (Promega, UK)
23.2
METHODS
For the following methods, we will describe the use of two specific mutant LHCGRs
that we have shown to undergo transactivation to rescue their respective functional
defects (
Rivero-M¨ ller et al., 2010
). The ligand-binding-deficient mutant LHCGR
(LHCGR
B
) contains a point mutation in the extracellular region, cysteine 22
to alanine (C22A), rendering it unable to bind the ligands LH or hCG (
Lee, Ji, &
Ji, 2002; Lee, Ji, Ryu, et al., 2002
). The signaling-deficient mutant LHCGR
(LHCGR
S
) contains the deletion valine 533-alanine 689, corresponding to trans-
membrane helices 6 and 7 (
Fig. 23.1
). The LHCGR
S
can bind ligand with the same
efficacy as the wild type (WT) LHCGR but is unable to activate G protein signaling,
as determined by measurement of generation of the second messenger cAMP.
Our rationale for choosing these specific LHCGR
B
and LHCGR
S
mutants was
driven by our aim to ultimately test whether transactivation of LHCGR could result in
the rescue of LHCGR function in
LhCGR
knockout (LuRKO) animals. We felt it was
imperative to use mutant LHCGRs that upon expression alone were completely devoid
of any ligand-binding or signaling activity. On this basis, we selected the LHCGR
B
C22A mutant initially as it had previously been shown by our collaborators to be
ligand-binding deficient but able to traffic to the cell surface (
Lee, Ji, & Ji, 2002;
Lee, Ji, Ryu, et al., 2002
) yet shown to undergo transactivation and functional rescue
when coexpressed with other signal-deficient or impaired receptors. Studies in our own
laboratories also confirmed the functional properties of this LHCGR
B
to be devoid of
ligand binding, and so it fulfilled our stringent criteria. We conducted functional assays
on several previously described LHCGR
S
and found the tested mutant LHCGRs
retained partial ligand-induced intracellular signaling and so for the purpose of our
in vivo
study could not be used. In order to be completely certain we would obtain
a LHCGR
S
totally devoid of ligand-induced signal generation, we took the approach
