A Simple and Powerful Flow Cytometric Method for the Simultaneous Determination of Multiple Parameters at G Protein‐Coupled Receptor Subtypes

Abstract

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.

[3H]UR‐MK136: A Highly Potent and Selective Radioligand for Neuropeptide Y Y1 Receptors

“…The resulting K i values of 1.5 nm for BIBP 3226 and 0.7 nm for pNPY are consistent with reported data (BIBP 3226: K i = 1.5, [6] 2.2, [3] 7.0, [1] 5.1 nm; [17] pNPY: K i = 0.7, [12] 0.5 nm [6] ). Furthermore, 5 b was used for the autoradiographic detection of Y 1 R in human MCF-7-Y 1 tumors, subcutaneously grown in nude mice.…”

“…The preparations of Dy-635-pNPY, Cy5-pNPY and Cy5- [K 4 ]hPP were reported previously. [12,14] Porcine NPY (pNPY) was prepared in the laboratory of Prof. Dr. C. Cabrele (Ruhr University, Bochum, Germany). Millipore water was used throughout for the preparation of buffers and HPLC eluents.…”

Application of the Guanidine–Acylguanidine Bioisosteric Approach to Argininamide‐Type NPY Y2 Receptor Antagonists

“…[23] To study receptor selectivity, all compounds were investigated in flow cytometric binding assays on human Y 1 , Y 4 , and Y 5 receptors using fluorescently labeled pNPY (Y 1 R, Y 5 R) or [K 4 ]hPP (Y 4 R) according to previously described methods (Table 2). [18,27,28] NPY Y 2 R binding and antagonism Two basic residues, Arg 33 and Arg 35, in the C terminus of NPY were identified to be crucial for high Y 2 R affinity. [29] The NPY mimetic argininamides related to BIIE0246 (1) also comprise two basic centers, namely the guanidino group in the arginine side chain and the tertiary amino nitrogen in the piperazine ring.…”

“…[a] Flow cytometric binding assays using fluorescently labeled pNPY or [K 4 ]hPP (Y 4 R) in CHO-hY 2 -K9-qi5-K9 cells, HEL-Y 1 cells, CHO-Y 4 , and HEC-1B-Y 5 cells, respectively (n = 3-5); [23,27,28] ND: not determined.…”

Identification of a Cyanine-Dye Labeled Peptidic Ligand for Y1R and Y4R, Based upon the Neuropeptide Y C-Terminal Analogue, BVD-15

“…Lys 4 (sCy5)-NPY was shown to be an agonist of Y1R, Y2R and Y4R receptors, and of utility in the development of FACS-based functional assays of complex cell-based assay systems. 17 Another approach has been to derive fluorescent or radiolabelled analogues of the Y1R arginamide antagonist series (BIBP3226, BIBO3304), 18 including pyridinium and cyanine based BIBP3226 derivatives suitable for fluorescent imaging. 19,20 .…”