Measuring GPCR signaling with a fast kinetic BRET assay

September 17, 2014

G-protein coupled receptors or GPCR's remain a very important subject of study due to their integral role in a variety of physiological processes.

Dr. Edward Dell | BMG LABTECH
Dr. EJ Dell
PhD, Sales Manager Northwest
BMG LABTECH USA

Therefore, continued efforts are being made to monitor the activity of GPCR's in order to identify new treatment options.

 

In a recent paper in the journal PLOS ONE the authors describe an approach based on bioluminescence resonance energy transfer or BRET to monitor dopamine receptor activation. As with other BRET approaches this assay monitors a protein-protein interaction which in this case is indicative of dopamine receptor activation. A binding peptide was linked to the recently engineered NanoLuc luciferase and a protein that binds to this peptide was linked to the Venus fluorophore. Treatment with agonist leads to association between the peptide and protein which brings NanoLuc into proximity of the Venus leading to a BRET signal.

 

To achieve detection of the BRET signal the authors used a POLARstar Omega plate reader from BMG LABTECH to detect light emitted by Venus and NanoLuc. Furthermore, they were able to perform detection with a 50 millisecond resolution which enabled detailed analysis of activation kinetics in response to dopamine as well as deactivation kinetics.

 

For more information on the POLARstar Omega and other microplate readers from BMG LABTECH click on the links.

 

Article citation: J.C. Octeau, et al. "G Protein Beta 5 Is Targeted to D2-Dopamine Receptor-Containing Biochemical Compartments and Blocks Dopamine-Dependent Receptor Internalization" PLoS One. 2014; 9(8)

go to top