title: Europium-labeled melanin-concentrating hormone analogues: ligands for measuring binding to melanin-concentrating hormone receptors 1 and 2.
abstract: We investigated the use of Eu3+ chelate-labeled analogues of melanin-concentrating hormone (MCH) as ligands for both human MCH receptors (MCHR1 and MCHR2). The analogues employed were Ala17 MCH, S36057 (Y-ADO-RC*MLGRVFRPC*W, where ADO=8-amino-3,6-dioxyoctanoyl and *=disulfide bond), and R2P (RC*MLGRVFRPC*Y-NH2). The peptides were readily labeled on the alpha-amino residue with the Eu3+ chelate of N1-(p-isothiocyanatobenzyl)-diethylenetriamine-N1,N2,N3,N3-tetraacetic acid and then purified by reverse-phase fast-performance liquid chromatography at neutral pH to maintain Eu3+ chelation. Both labeled Ala17 MCH and S36057 had high affinity for MCHR1 ( Kd = 0.37 and 0.059nM, respectively) while Eu3+ -labeled S36057 and R2P had high affinity for MCHR2 ( Kd = 0.16 and 0.10nM, respectively). Labeled Ala17 MCH had little demonstrable binding affinity for MCHR2. Eu3+ -labeled S36057 and R2P were full agonists at MCHR1 when assessed by measurement of agonist-stimulated GTPgamma(35)S binding. Competition binding experiments with both MCHR isoforms, a series of previously characterized alanine scan MCH analogues, and a recently identified nonpeptide MCHR1-selective antagonist T-226296 confirmed the expected receptor selectivity. These studies further extend the utility of Eu3+ chelate time-resolved fluorescence for the development of high-sensitivity, nonradioactive receptor binding assays and demonstrate the need to select the optimal ligand for labeling.
authors.affiliation: Endocrine Discovery Research, Lilly Research Laboratories, Eli Lilly and Co., DC 0545, Indianapolis, IN 46285, USA.
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Gao
authors.forename: Chiun-King
authors.initials: CK
authors.lastname: Hsu
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Heinz
authors.forename: John
authors.initials: J
authors.lastname: Morin
authors.forename: Yuguang
authors.initials: Y
authors.lastname: Shi
authors.forename: Nikhil K
authors.initials: NK
authors.lastname: Shukla
authors.forename: David L
authors.initials: DL
authors.lastname: Smiley
authors.forename: Jie
authors.initials: J
authors.lastname: Xu
authors.forename: Boyu
authors.initials: B
authors.lastname: Zhong
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Slieker
mesh_terms: D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D005063:Europium / Q000737:chemistry*; D005453:Fluorescence; D006801:Humans; D008024:Ligands; D008543:Melanins / Q000737:chemistry / Q000378:metabolism*; D009942:Organometallic Compounds / Q000737:chemistry; D011485:Protein Binding; D011869:Radioligand Assay; D011979:Receptors, Pituitary Hormone / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling
chemical_list: D004220:Disulfides; D008024:Ligands; D008543:Melanins; D009942:Organometallic Compounds; D011979:Receptors, Pituitary Hormone; C475854:europium(III) chelate-2,2',2'',2'''-((4-((4-isothiocyanatophenyl)ethynyl)pyridine-2,6-diyl)bis(methylenenitrilo))tetrakis(acetic acid); C092274:melanin-concentrating hormone receptor; D005063:Europium