title: 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 Eu3+. The relatively simple synthesis yields ligands with acceptable binding characteristics similar to isotopically labeled derivatives. The binding affinity (Kd) of labeled Eu-EGF to human A431 epidermal carcinoid cells was 3.6 +/- 1.2 nM, similar to the reported Kd values of EGF, whereas the Kd 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 Kd 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 Ca2+ influx with NT and Eu-NT in CHO-NT1 cells was similar, but the respective K0.5 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.
authors.affiliation: Department of Biological Regulation, Weizmann Institute of Science, Rehovot, 76100, Israel.
authors.forename: Ohad
authors.initials: O
authors.lastname: Mazor
authors.forename: Marc
authors.initials: M
authors.lastname: Hillairet de Boisferon
authors.forename: Alain
authors.initials: A
authors.lastname: Lombet
authors.forename: Anne
authors.initials: A
authors.lastname: Gruaz-Guyon
authors.forename: Batya
authors.initials: B
authors.lastname: Gayer
authors.forename: Delphine
authors.initials: D
authors.lastname: Skrzydelsky
authors.forename: Fortune
authors.initials: F
authors.lastname: Kohen
authors.forename: Patricia
authors.initials: P
authors.lastname: Forgez
authors.forename: Avigdor
authors.initials: A
authors.lastname: Scherz
authors.forename: William
authors.initials: W
authors.lastname: Rostene
authors.forename: Yoram
authors.initials: Y
authors.lastname: Salomon
mesh_terms: D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D006224:Cricetinae; D004815:Epidermal Growth Factor / Q000737:chemistry*; D066246:ErbB Receptors / Q000032:analysis; D005063:Europium / Q000737:chemistry*; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D051379:Mice; D015335:Molecular Probes / Q000737:chemistry; D009496:Neurotensin / Q000737:chemistry*; D011956:Receptors, Cell Surface / Q000032:analysis*; D018028:Receptors, Neurotensin / Q000032:analysis; D014407:Tumor Cells, Cultured
chemical_list: D015335:Molecular Probes; D011956:Receptors, Cell Surface; D018028:Receptors, Neurotensin; D009496:Neurotensin; D005063:Europium; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D002118:Calcium