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

Xiaoying Gao, Chiun King Hsu, Lawrence J. Heinz, John Morin, Yuguang Shi, Nikhil K. Shukla, David L. Smiley, Jie Xu, Boyu Zhong, Lawrence J. Slieker

Research output: Contribution to journalArticlepeer-review

14 Scopus citations

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 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 (Kd=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.

Original languageEnglish (US)
Pages (from-to)187-195
Number of pages9
JournalAnalytical Biochemistry
Volume328
Issue number2
DOIs
StatePublished - May 15 2004
Externally publishedYes

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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