A TIME-RESOLVED FLUOROMETRIC ASSAY FOR GALANIN RECEPTORS

A time-resolved fluorometric (TRF) assay was developed for human galan in receptors, subtype 1 (hGalR1), The galanin peptide was labeled with a europium chelate, Eu3+-DTTA. Ligand binding was conducted in D98/Ra ji cells expressing recombinant human GalR1 (hGalR1-D98/Raji) in monol ayers in 96-well plates or in crude membrane preparations in 96-well f ilter plates. Bound ligand was quantitated by dissociation-enhanced ti me-resolved fluorometric measurement of Eu3+. Eu(3+-)galanin bound to hGalR1 in a saturable manner with K-d values of 2.9 +/- 0.5 nM in whol e cell assays and 0.23 +/- 0.03 nM in membrane assays, The affinities of Eu3+-galanin for hGalR1 in both the whole cell and membrane assays correlated very well with those of I-125-galanin. In addition, the bou nd Eu3+-galanin could be competitively displaced from the receptors by galanin peptide antagonists C7, M15, M35, and M40. In hGalR1-expressi ng 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 li braries. The sensitivity of this assay is comparable with that of I-12 5-galanin. The advantages of this assay include elimination of radioac tivity, stability, low cost, speed, and amenability for automation.