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Journal of Biomolecular Screening
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A Time-Resolved Fluorometric Assay for Galanin Receptors

Jie Liu

Department of Drug Discovery Research, Pharmacopeia, Inc., CN5350, Princeton, NJ 08543-5350

Michael Gallagher

Department of Drug Discovery Research, Pharmacopeia, Inc., CN5350, Princeton, NJ 08543-5350

Robert A. Horlick

Department of Drug Discovery Research, Pharmacopeia, Inc., CN5350, Princeton, NJ 08543-5350

Alan K. Robbins

Department of Drug Discovery Research, Pharmacopeia, Inc., CN5350, Princeton, NJ 08543-5350

Maria L. Webb

Department of Drug Discovery Research, Pharmacopeia, Inc., CN5350, Princeton, NJ 08543-5350

A time-resolved fluorometric (TRF) assay was developed for human galanin receptors, subtype 1 (hGalR1). The galanin peptide was labeled with a europium chelate, Eu3+-DTTA. Ligand binding was conducted in D98/Raji cells expressing recombinant human GalR1 (hGalRl-D98/Raji) in monolayers in 96-well plates or in crude membrane preparations in 96-well filter plates. Bound ligand was quantitated by dissociation-enhanced time-resolved fluorometric measurement of Eu3+. Eu3+-galanin bound to hGalRl in a saturable manner with Kd values of 2.9 ± 0.5 nM in whole cell assays and 0.23 ± 0.03 nM in membrane assays. The affinities of Eu3+-galanin for hGalRl in both the whole cell and membrane assays correlated very well with those of 125I-galanin. In addition, the bound Eu3+-galanin could be competitively displaced from the receptors by galanin peptide antagonists C7, M15, M35, and M40. In hGalRl-expressing 293EBNA cells, Eu3+-galanin stimulated an increase in intracellular Ca2+ concentration with a similar EC50 value as unlabeled galanin (4.5 ± 0.5 nM versus 4.2 ± 0.3 nM), indicating that Eu3+ labeling did not affect the functional activity of galanin. The galanin TRF assay was employed in high throughput screening of combinatorial chemical libraries. The sensitivity of this assay is comparable with that of 125I-galanin. The advantages of this assay include elimination of radioactivity, stability, low cost, speed, and amenability for automation.

Journal of Biomolecular Screening, Vol. 3, No. 3, 199-206 (1998)
DOI: 10.1177/108705719800300306
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