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2008
). Sterols such as cholesterol have also been noted to influence some GPCRs
(
Oates & Watts, 2011
), and their presence can be modulated via, for example, the
use of cyclodextrins (
Oates et al., 2012
).
Due to difficulties in modulating bilayer lipid composition in living cells, it is
easiest to perform these studies
in vitro
. Such studies necessitate purification of ac-
tive GPCRs, a process that has proved challenging. GPCRs are often unstable in de-
tergent solution, which has been one of the main barriers to structural determination
(
Bill et al., 2011
). However, advances made with modern detergents, thermostabi-
lized mutants, and rapid purification strategies enable the production of a number
of purified, active receptors (
Zhao & Wu, 2012
).
Once the receptor has been purified, it must be reconstituted into a defined lipid
environment. A number of different methods exist including the use of liposomes,
nanodiscs (
Bayburt & Sligar, 2010
), and Lipodisqs (
Orwick et al., 2012
). In this
chapter, we will focus on the use of liposomes as it is possible that the use of small
lipid discs will result in a high local GPCR concentration and hence will produce
artifactual interactions. Once reconstituted, the ability of GPCRs to dimerize
must be quantified. Although a number of techniques can be applied, our laboratory,
and this chapter, focuses on the use of FRET. This technique can be used to indicate
protein-protein interactions as the resonance energy transfer is generally limited to a
distance of a few nanometers. However, FRET should be used judiciously and a
number of controls must be performed to ensure that results are meaningful; such
controls will be discussed in the succeeding text.
Here, we describe our approaches to GPCR reconstitution into liposomes con-
taining defined lipid mixtures and the subsequent measurement and analysis of
FRET to quantify the interaction between receptors under different lipid conditions.
These techniques should be transferrable to any GPCR and also a variety of other
integral or membrane-associated proteins.
18.1
MATERIALS AND METHOD
18.1.1
Purification of GPCRs
GPCRs can be purified using a number of expression systems including
E. coli
,
baculovirus/insect cells, mammalian cells, yeast, and
in vitro
translation systems.
The advantages and disadvantages of such systems are described elsewhere
(
Tapaneeyakorn, Goddard, Oates, Willis, &Watts, 2011
). Our group and others have
optimized expression and purification of neurotensin receptor 1 (NTS1), which binds
neurotensin (NT) (
Attrill, Harding, Smith, Ross, &Watts, 2009; Grisshammer, 2009;
Harding et al., 2007
). We use a fusion protein (FP) in which NTS1 has an N-terminal
maltose-binding protein domain and a C-terminal thioredoxin domain with decahis-
tidine tag added. Tobacco etch virus (TeV) protease sites are present at the junctions
to remove the tags postpurification. In the case of fluorescently tagged variants,
CFP or YFP is appended to the C-terminus of NTS1 (
Harding et al., 2009
) before
the TeV site. Receptors are initially purified from
E. coli
using immobilized metal
