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

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 α-amino residue with the Eu ³⁺ chelate of N¹-(p-isothiocyanatobenzyl)- diethylenetriamine-N¹,N²,N³,N ³-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 (K_d=0.37 and 0.059nM, respectively) while Eu ³⁺-labeled S36057 and R2P had high affinity for MCHR2 (K _d=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 GTPγ³⁵S 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.