Melanocortin 4 receptors interact with antimicrobial frog peptide analogues q Ernest U. Do a , Eun Bae Jo a , Gyu Choi b , Long Zhu Piao b , Jaekyoon Shin b , Min-Duk Seo c , Su-Jin Kang c , Bong-Jin Lee c , Kang Ho Kim d , Jae Bum Kim d , Su-il Kim a, * a School of Agricultural Biotechnology, Seoul National University, San 56-1 Sillim-dong, Gwanak-gu, Seoul 151-742, Republic of Korea b School of Medicine, Department of Molecular Cell Biology, Sungkyunkwan University, 300 Chunchun-dong, Jangan-gu, Suwon 440-746, Republic of Korea c College of Pharmacy, Seoul National University, San 56-1 Sillim-dong, Gwanak-gu, Seoul 151-742, Republic of Korea d Department of Biological Sciences, College of Natural Sciences, Seoul National University, San 56-1 Sillim-dong, Gwanak-gu, Seoul 151-747, Republic of Korea Received 28 February 2006 Available online 23 March 2006 Abstract We have developed fluorescence polarization (FP) assays of human melanocortin 4 receptor (MC4R) in 384-well microtiter plates using TAMRA-NDP-MSH as a tracer. The rank order of potency of agonists and antagonists agrees well relative to the published assays: SHU9119 > MTII > NDP aMSH > aMSH. We have screened libraries of Korean plant extracts and frog peptide analogues in search of MC4R ligands using FP assays and cell-based CRE luciferase reporter assays. We report that FLGFLFKVASK, FLGWLFKVASK, FLGALFKWASK, and FLGWLFKWASK are the peptide analogues, which bind to human MC4R receptor with good affinity in vitro. FLGWLFKVASK and FLGWLFKWASK stimulated CRE-driven reporter gene via MC4R. In luciferase reporter assays, they possess the pharmacological and functional profiles of full agonists. We demonstrate the interaction of MC4R with 11- residue antimicrobial peptides derived from the Korean frog, Rana rugosa. The results suggest that MC4R interacts promiscuously with bioactive analogues of antimicrobial peptide, gaegurin-5. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Fluorescence polarization; High-throughput screening; Human melanocortin 4 receptors; Agonists and antagonists; Rank order of potency; Gaegurin The melanocortin 4 receptor (MC4R) has been a focus of recent interest as a critical regulator of energy homeosta- sis and food intake, and as a mediator of antipyretic and hyperthermic actions [1–3]. MC4R and its mRNA-express- ing cells are predominantly located in the central nervous systems and MC4R is the principal melanocortin receptor subtype of the cortex, hypothalamus, brain stem, and spinal cord [4,5]. MC4R agonists decrease food intake and MC4R antagonists increase food intake after central administration [1,6–12]. MC4R has been implicated in other physiological processes such as release of insulin [13], luteinizing hormone and prolactin [14,15], and obesity [5,10,13,16,17]. MC4R is important for mediation of the effects of mela- nocyte-stimulating hormone (MSH) peptides [1], agouti protein [18], and agouti-related peptides (AgRP) [19,20]. Intracerebroventricular (ICV) injection of MTII, a MC4R agonist, reduced food intake up to 4 h and caused a large increase in water intake during the first 4 h after 0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.03.082 q Abbreviations: AgRP, agouti-related peptides; cAMP, cyclic 3 0 ,5 0 - adenosine monophosphate; DMSO, dimethyl sulfoxide; FP, fluorescence polarization; GPCRs, G-protein coupled receptors; ICV, intracerebro- ventricular; MC4R, melanocortin 4 receptor; MSH, melanocyte stimulat- ing hormone; NDP, Nle 4 ,D-Phe 7 ; HTS, high-throughput screening; mP, milli-polarization; PEG, polyethylene glycol; MTII, (acetyl-Nle 4 ,Asp 5 , D-Phe 7 ,Lys 10 )-cyclo-aMSH (4-10) amide (melanotan II); NPFF, neuro- peptide FF; SHU9119, (acetyl-Nle 4 ,Asp 5 ,D-2-Nal 7 ,Lys 10 )-cyclo-aMSH (4-10) amide; TAMRA, 5-carboxymethylrhodamine; GGN, gaegurin. * Corresponding author. Fax: +822 873 2039. E-mail address: sikim@snu.ac.kr (S. Kim). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 343 (2006) 1094–1100 BBRC