Biology Reference
In-Depth Information
CHAPTER
9
Eug ´ nie Goupil
*
, St ´ phane A. Laporte
*
,{
, and Terence E. H ´bert
*
*
A Simple Method to Detect
Allostery in GPCR Dimers
Department of Pharmacology, McGill University, Montre´al, Que´bec, Canada
{
Department of Medicine, McGill University, Montre´al, Que´bec, Canada
CHAPTER OUTLINE
Introduction and Rationale....................................................................................... 166
9.1 Materials......................................................................................................... 169
9.2 Methods .......................................................................................................... 170
9.2.1 Cell Culture and Transfection .......................................................... 170
9.2.2 Radioligand Binding and Dissociation Kinetics.................................. 170
9.2.3 Analysis and Interpretation of Results .............................................. 172
9.3 Discussion....................................................................................................... 174
Summary ................................................................................................................ 176
Acknowledgments ................................................................................................... 176
References ............................................................................................................. 176
Abstract
G protein-coupled receptors (GPCRs) represent one of the largest families of cell
surface receptors as key targets for pharmacological manipulation. G proteins have
long been recognized as allosteric modulators of GPCR ligand binding. More re-
cently, small molecule allosteric modulators have now been widely characterized
for a number of GPCRs, and some are now used clinically. Many studies have also
underscored the importance of GPCR dimerization or higher-order oligomerization
in the control of the physiological responses they modulate. Thus, allosterism can
also, between monomer equivalents in the context of a dimer, oligomer, or receptor
mosaic, influence signaling pathways downstream. It therefore becomes essential to
characterize both small molecule allosteric ligands and allosteric interactions be-
tween receptors modulated by canonical orthosteric ligands, in a pathway-specific
manner. Here, we describe a simple, radioligand-binding method, which is designed
to probe for allosteric modulation mediated by any GPCR interactor, from small mol-
ecules to interacting proteins. It can also detect allosteric asymmetries within a
