Biology Reference
In-Depth Information
CHAPTER
12
Biochemical Assay of G
Protein-Coupled Receptor
Oligomerization: Adenosine
A
1
and Thromboxane A
2
Receptors Form the Novel
Functional Hetero-oligomer
Natsumi Mizuno
*
, Tokiko Suzuki
{
, Yu Kishimoto
*,
and Noriyasu Hirasawa
*
*
Department of Pharmacotherapy of Life-style Related Disease, Graduate School of
Pharmaceutical Sciences, Tohoku University, Sendai, Japan
{
Department of Cellular Signaling, Graduate School of Pharmaceutical Sciences, Tohoku
University, Sendai, Japan
CHAPTER OUTLINE
Introduction ............................................................................................................ 214
12.1 Detection of GPCR Oligomerization...................................................................216
12.1.1 Coimmunoprecipitation............................................................... 216
12.1.1.1 Coimmunoprecipitation of the GPCR Oligomer.................... 216
12.1.1.2 Protocol for Coimmunoprecipitation.................................... 216
12.1.1.3 Coimmunoprecipitation Results .......................................... 218
12.1.2 BRET
2
Assay.............................................................................. 218
12.1.2.1 BRET
2
Assay to Analyze GPCR Oligomerization .................. 218
12.1.2.2 BRET
2
Saturation Assay..................................................... 219
12.1.2.3 Competitive BRET
2
Assay .................................................. 219
12.2 Measurement of the Signaling Pathway ............................................................220
12.2.1 Cyclic AMP Assay ....................................................................... 220
12.2.2 Extracellular signal-regulated Kinase1/2 Assay.............................. 222
12.2.3 Effect of the Coexpression of Receptors on the Signaling Pathway .. 222
12.3 Materials and Methods....................................................................................223
12.3.1 Materials ................................................................................... 223
12.3.2 Construction of Plasmids ............................................................ 223
12.3.3 Cell Culture and Transfection ...................................................... 223
12.3.4 Western Blot Analysis ................................................................. 224
Conclusion ............................................................................................................. 224
References ............................................................................................................. 224
