Biology Reference
In-Depth Information
CHAPTER
18
Alan D. Goddard * , Patricia M. Dijkman { , Roslin J. Adamson { , and Anthony Watts {
*
Lipid-Dependent GPCR
Dimerization
School of Life Sciences, University of Lincoln, Lincoln, United Kingdom
{ Biomembrane Structure Unit, Department of Biochemistry, University of Oxford, Oxford,
United Kingdom
CHAPTER OUTLINE
Introduction ............................................................................................................ 342
18.1 Materials and Method .....................................................................................343
18.1.1 Purification of GPCRs ................................................................. 343
18.1.2 Labeling Strategies for FRET ....................................................... 344
18.1.2.1 Protocol for Site-Directed Fluorophore Labeling
of Cysteines....................................................................... 345
18.1.3 Production of Liposomes ............................................................. 347
18.1.3.1 Preparing the Lipid ............................................................ 347
18.1.3.2 Sizing Liposomes: Sonication Versus Extrusion ................... 347
18.1.4 Reconstitution............................................................................ 348
18.1.4.1 Protocol for Reconstitution Using Bio-beads ....................... 349
18.2 FRET Measurements and Analysis ....................................................................351
18.3 Interpreting Results.........................................................................................354
Summary ................................................................................................................ 355
References ............................................................................................................. 355
Abstract
It has been widely demonstrated that G protein-coupled receptors (GPCRs) can form
dimers both in vivo and in vitro , a process that has functional consequences. These
receptor-receptor interactions take place within a phospholipid bilayer, yet, gener-
ally, little is known of the requirements for specific lipids that mediate the dimeriza-
tion process. Studying this phenomenon in vivo is challenging due to difficulties in
modulating the lipid content of cell membranes. Therefore, in this chapter, we de-
scribe techniques for reconstitution of GPCRs into model lipid bilayers of defined
composition. The concentrations of specific lipids and sterols can be precisely
 
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