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

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

  • Anal Biochem. 2004 May 15;328(2):187-95. doi: 10.1016/j.ab.2004.01.017.
Xiaoying Gao 1 Chiun-King Hsu Lawrence J Heinz John Morin Yuguang Shi Nikhil K Shukla David L Smiley Jie Xu Boyu Zhong Lawrence J Slieker
Affiliations

Affiliation

  • 1 Endocrine Discovery Research, Lilly Research Laboratories, Eli Lilly and Co., DC 0545, Indianapolis, IN 46285, USA.
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.

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