Development and validation of algorithms for measuring G-protein coupled receptor activation in cells using the LSC-based imaging cytometer platform.
BACKGROUND
A cell-based assay system (Transfluor) has been developed for measurement of G-protein coupled receptor (GPCR) activity by using cells transfected to express a fusion protein of arrestin plus green fluorescent protein (GFP) and the target GPCR. Upon agonist stimulation, the arrestin-GFP translocates to and binds the activated GPCR at the plasma membrane. The receptor/arrestin-GFP complexes then localize in clathrin-coated pits and/or intracellular vesicles. This redistribution of arrestin-GFP into condensed fluorescent spots is useful for visually monitoring the active status of GPCRs and its quantitation is possible with certain types of digital image analysis systems.


METHODS
We designed two lines of image processing algorithms to carry out quantitative measurement of the arrestin-GFP movement on an inverted version of laser scanning cytometry (iCyte) as an imaging platform. We used a cell line expressing arrestin-GFP and the wild-type beta2-adrenergic receptor or a modified version of this receptor with enhanced affinity for arrestin. Each cell line was challenged with various concentrations of agonist.


RESULTS
A dose-dependent signal was measured and half-maximal effective concentration values were obtained that agreed well with results determined by other methods previously reported.


CONCLUSIONS
The results indicate that the combination of Transfluor, iCyte, and our algorithms is suitable for robust and pharmacologically relevant GPCR ligand exploration.
Ozawa
Kazuo
K
Olympus Corporation, Bioscience Division, Tokyo, Japan. kazuo1_ozawa@ot.olympus.co.jp
Hudson
Christine C
CC
Wille
Kirsten R
KR
Karaki
Sachiko
S
Oakley
Robert H
RH
D000465:Algorithms; D045744:Cell Line, Tumor; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000378:metabolism; D006801:Humans; D007090:Image Interpretation, Computer-Assisted / Q000379:methods* / Q000592:standards; D047569:Laser Scanning Cytometry / Q000379:methods* / Q000592:standards; D008024:Ligands; D043562:Receptors, G-Protein-Coupled / Q000032:analysis* / Q000378:metabolism* / Q000648:ultrastructure; D012680:Sensitivity and Specificity
D008024:Ligands; D043562:Receptors, G-Protein-Coupled; D049452:Green Fluorescent Proteins