Biology Reference
In-Depth Information
controlled in these liposomes, as well as maintaining an appropriate lipid-protein
ratio to avoid artifactual interactions. Receptor dimerization in this system is mon-
itored via F¨rster resonance energy transfer (FRET), which requires the use of fluo-
rescently labeled receptors. We therefore also include protocols for labeling with
appropriate fluorophores and determining the apparent FRET efficiency, a measure-
ment of the extent of receptor dimerization. Understanding the lipid dependence of
GPCR dimerization will be key in understanding how this process is regulated in the
dynamic heterogeneous environment of the cell membrane.
INTRODUCTION
All organisms rely on membrane proteins to detect and respond to changes in their
external environment. G protein-coupled receptors (GPCRs) represent the largest
family of mammalian cell-surface receptors, with
800 identified in humans,
constituting
1% of the genome (
Vassilatis et al., 2003
). GPCRs generally bind
an extracellular ligand and transduce, via a conformational change, this signal to
an intracellular heterotrimeric G protein consisting of G
a
,G
b
, and G
g
subunits.
Exchange of GDP for GTP on the G
a
subunit results in activation of various signal-
ing cascades bringing about cellular responses (
Oldham & Hamm, 2008
). GPCRs
are involved in a plethora of physiological processes and hence are important drug
targets—it is estimated that
30% of pharmaceutical drugs target these receptors
(
Jacoby, Bouhelal, Gerspacher, & Seuwen, 2006
).
An emerging theme over the last decade has been the ability of GPCRs to form
dimers or higher-order oligomers with either the same GPCR or other members of
this family. Such dimerization has been shown to affect both ligand binding and
signaling (
Ciruela et al., 2010
); indeed, constitutive dimerization of class C GPCRs
is essential for their activity. While GPCR dimerization has been demonstrated
in
vitro
and
in vivo
, little is known of the lipid-dependence of these interactions.
Membrane proteins do not exist in isolation but are influenced, both chemically
and physically, by lipid, protein, and other components of the membrane including
annular lipids, which interact directly with the proteins, and factors arising from the
bulk of the bilayer such as lateral pressure. Lipid bilayers have a number of char-
acteristics that are important for protein activity and are strongly influenced by their
lipid composition. Membrane thickness is dependent upon the nature of the hydro-
carbon chain of the lipid. The charge on the membrane is influenced by the head
groups of the lipids within the bilayer. Additionally, the membrane may be asym-
metric, that is, it may not have the same lipid composition within each leaflet
(
van Meer, 2011
). Finally, lipid membranes adopt different phases that are influ-
enced by the temperature of the environment and the lipid composition of the mem-
brane. Bilayers are not necessarily homogenous across their surface and may form
microdomains such as lipid rafts or caveolae, and it has been demonstrated that
some GPCRs and G proteins localize to such regions (
Patel, Murray, & Insel,
