Biology Reference
In-Depth Information
CHAPTER
8
Bioluminescence
Resonance Energy Transfer
Methods to Study
G Protein-Coupled
Receptor-Receptor Tyrosine
Kinase Heteroreceptor
Complexes
Dasiel O. Borroto-Escuela
*
, Marc Flajolet
{
, Luigi F. Agnati
{
, Paul Greengard
{
, and
Kjell Fuxe
*
*
Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden
{
Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, USA
{
IRCCS Lido, Venice, Italy
CHAPTER OUTLINE
Introduction ............................................................................................................ 142
8.1 The Method Principle ....................................................................................... 143
8.2 Setting Up a BRET Assay .................................................................................. 146
8.3 Methods and Detailed Protocols........................................................................ 148
8.3.1 Protocol 1: Saturation Assay............................................................ 151
8.3.2 Protocol 2: Competition Assays........................................................ 155
8.3.3 Protocol 3: Kinetics and Dose-response Assays................................. 156
8.4 Discussion and Note on Critical Parameters ...................................................... 159
Acknowledgments ................................................................................................... 161
References ............................................................................................................. 162
Abstract
A large body of evidence indicates that G protein-coupled receptors (GPCRs) and
receptor tyrosine kinases (RTKs) can form heteroreceptor complexes. In these com-
plexes, the signaling from each interacting protomer is modulated to produce an in-
tegrated and therefore novel response upon agonist(s) activation. In the GPCR-RTK
