Biology Reference
In-Depth Information
Abstract
Oligomerization is one of several mechanisms that can regulate the activity of G
protein-coupled receptors (GPCRs), but little is known about the structure of GPCR
oligomers. Crystallography and NMR are the only methods able to reveal the details
of receptor-receptor interactions at an atomic level, and several GPCR homodimers
already have been described from crystal structures. Two clusters of symmetric in-
terfaces have been identified from these structures that concur with biochemical data,
one involving helices I, II, and VIII and the other formed mainly by helices V and VI.
In this chapter, we describe the protocols used in our laboratory for the crystallization
of rhodopsin and the
2-AR). For bovine rhodopsin, we de-
veloped a new purification strategy including a (NH
4
)
2
SO
4
-induced phase separation
that proved essential to obtain crystals of photoactivated rhodopsin containing par-
allel dimers. Crystallization of native bovine rhodopsin was achieved by the classic
vapor-diffusion technique. For
b
2-adrenergic receptor (
b
b
2-AR, we developed a purification strategy based on
previously published protocols employing a lipidic cubic phase to obtain diffracting
crystals of a
b
2-AR/T4-lysozyme chimera bound to the antagonist carazolol.
Abbreviations
CHS
cholesterol hemisuccinate
dodecyl-
b
-
D
-maltoside
DDM
FRAP
fluorescence recovery after photobleaching
GPCR
G protein-coupled receptor
LCP
lipidic cubic phase
MES
2-(
N
-morpholino) ethanesulfonic acid
NG
nonyl-
b
-
D
-glucoside
ROS
rod outer segment(s)
T4L
T4 lysozyme
b
2-AR
b
2-adrenergic receptor
INTRODUCTION
After a decade of heated debate, a large body of experimental data now supports the
notion that G protein-coupled receptors (GPCRs) can form physiologically relevant
oligomers (
Milligan, 2008
). Therefore, GPCR oligomerization interface(s) are po-
tential targets for allosteric drugs to modulate GPCR function and high-resolution
structures of GPCR oligomers are highly sought as templates for structure-based
drug design. The first direct structural evidence of GPCR oligomerization was
obtained in our laboratory by atomic-force microscopy, showing a paracrystalline
arrangement of bovine rhodopsin in native membranes (
Fotiadis et al., 2003
). Later,
parallel rhodopsin dimers were observed in crystals solved by electron crystallogra-
phy at 5.5
˚
(
Ruprecht, Mielke, Vogel, Villa, & Schertler, 2004
) and by X-ray
