Europium-Labeled Epidermal Growth Factor and Neurotensin: Novel Probes for Receptor-Binding Studies

Abstract

We investigated the possibility of labeling two biologically active peptides, epidermal growth factor (EGF) and neurotensin (NT), with europium (Eu)-diethylenetriaminepentaacetic acid. More specifically, we tested them as probes in studying receptor binding using time-resolved fluorescence of Eu 3؉ . The relatively simple synthesis yields ligands with acceptable binding characteristics similar to isotopically labeled derivatives. The binding affinity (K d ) of labeled Eu-EGF to human A431 epidermal carcinoid cells was 3.6 ؎ 1.2 nM, similar to the reported K d values of EGF, whereas the K d of Eu-NT to human HT29 colon cancer cells (7.4 ؎ 0.5 nM) or to Chinese hamster ovary (CHO) cells transfected with the high-affinity NT receptor (CHO-NT1) were about 10-fold higher than the K d values of NT. The bioactivity of the Eu-labeled EGF as determined by stimulation of cultured murine D1 hematopoietic cell proliferation was nearly the same as that obtained with native EGF. The maximal stimulation of Ca 2؉ influx with NT and Eu-NT in CHO-NT1 cells was similar, but the respective K 0.5 values were 20 pM and 1 nM, corresponding to differences in the binding affinities previously described. The results of these studies indicate that Eu labeling of peptide hormones and growth factor molecules ranging from 10 3 to 10 5 Da can be conveniently accomplished. Importantly, the Eu-labeled products are stable for approximately 2 years and are completely safe for laboratory use compared to the biohazardous radioligands. Thus, Eu-labeled peptides present an attractive alternative for commonly used radiolabeled ligands in biological studies in general and in receptor assays in particular.

Modulation of response to estrogens in cultured human female bone cells by a non-calcemic Vitamin D analog: changes in nuclear and membranal binding

“…Cells were incubated with the steroid protein conjugates (E 2 -Ov) [14] labeled with Eu at 4 • C, and binding was performed and analyzed by time-resolved fluorescence using an Arcus time-resolved fluorometer (Wallac, Turku, Finland) as described before [15].…”

Role of putative membrane receptors in the effect of androgens on human vascular cell growth

“…Cells (4 10 5 cells/well) cultured in 24-well plates for 48 h were washed once with ice-cold binding medium (Dulbecco's modified Eagles' medium+0·1% BSA and HEPES 25 mM, pH 7·4) using reaction conditions as described previously (Mazor et al 2002). Subsequently, cells were incubated for 90 min at 4 C with the steroid protein conjugates (E 2 -6-CMO ovalbumin, T-3-CMO-BSA; Kohen et al 1975; 10 µM/well).…”

Lanthanide-based luminescent assays for ligand-receptor interactions

“…Many recent reports (Gao et al, 2004;Handl et al, 2004;Inglese et al, 1998;Mazor et al, 2002;Takeuchi et al, 1995) have described the development of Eu-labeled ligands that can be competed off by compound libraries in competitive binding assays ( Table 1). Most of these reports have described the use of lanthanide labeled ligands in a traditional competitive binding assay format.…”