A simple and powerful flow cytometric method for the simultaneous determination of multiple parameters at G protein-coupled receptor subtypes.
The quantification of pharmacological parameters at G protein-coupled receptors (GPCRs) is indispensable in drug research but costly and time-consuming when conventional methods are sequentially applied. With neuropeptide Y (NPY) Y(1), Y(2), and Y(5) receptors as model systems, a homogenous flow cytometric method for the simultaneous determination of the affinity, selectivity, and activity of GPCR ligands was developed. Mixtures of cells expressing the receptors of interest and cyanine-labeled NPY as a universal fluorescent Y(1), Y(2), and Y(5) receptor agonist were used. Calcium mobilization was measured in different channels with the aid of fluo-4 and fura red. A combination of dye-loaded HEL-Y(1) and CHO-Y(2)-Galpha(qi5) cells with unloaded HEC-1B-Y(5) cells allowed the simultaneous determination of Y(1), Y(2), and Y(5) receptor selectivity preceded by the Y(1) and Y(2) receptor-mediated response with one and the same sample. The data are in good agreement with those determined by radioligand binding and spectrofluorimetry. The convenient, robust, and inexpensive multiparametric procedure offers a broad range of applications in the pharmacological characterization of GPCR ligands.
Schneider
Erich
E
University of Regensburg, Institute of Pharmacy, 93040 Regensburg, Germany.
Mayer
Matthias
M
Ziemek
Ralf
R
Li
Liantao
L
Hutzler
Christoph
C
Bernhardt
Günther
G
Buschauer
Armin
A
D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D045744:Cell Line, Tumor; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D015394:Molecular Structure; D009478:Neuropeptide Y / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011869:Radioligand Assay; D043562:Receptors, G-Protein-Coupled / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D017476:Receptors, Neuropeptide Y / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D013552:Swine
D005456:Fluorescent Dyes; D009478:Neuropeptide Y; D043562:Receptors, G-Protein-Coupled; D017476:Receptors, Neuropeptide Y; C097212:neuropeptide Y-Y1 receptor; C095889:neuropeptide Y2 receptor; C100568:neuropeptide Y5 receptor; D002118:Calcium