Europium-labeled melanin-concentrating hormone analogues: ligands for measuring binding to melanin-concentrating hormone receptors 1 and 2

Abstract We investigated the use of Eu 3+ chelate-labeled analogues of melanin-concentrating hormone (MCH) as ligands for both human MCH receptors (MCHR1 and MCHR2). The analogues employed were Ala 17 MCH, S36057 (Y-ADO-RC*MLGRVFRPC*W, where ADO=8-amino-3,6-dioxyoctanoyl and *=disulfide bond), and R2P (RC*MLGRVFRPC*Y-NH 2 ). The peptides were readily labeled on the α-amino residue with the Eu 3+ chelate of N 1 -( p -isothiocyanatobenzyl)-diethylenetriamine- N 1 , N 2 , N 3 , N 3 -tetraacetic acid and then purified by reverse-phase fast-performance liquid chromatography at neutral pH to maintain Eu 3+ chelation. Both labeled Ala 17 MCH and S36057 had high affinity for MCHR1 ( K d =0.37 and 0.059 nM, respectively) while Eu 3+ -labeled S36057 and R2P had high affinity for MCHR2 ( K d =0.16 and 0.10 nM, respectively). Labeled Ala 17 MCH had little demonstrable binding affinity for MCHR2. Eu 3+ -labeled S36057 and R2P were full agonists at MCHR1 when assessed by measurement of agonist-stimulated GTPγ 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 Eu 3+ 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.