Molecular cloning of the bullfrog kisspeptin receptor GPR54 with high sensitivity to Xenopus kisspeptin Jung Sun Moon a,1 , Yeo Reum Lee a,1 , Da Young Oh a , Jong Ik Hwang a , Ju Yeon Lee b , Jae Il Kim b , Hubert Vaudry c , Hyuk Bang Kwon d , Jae Young Seong a, * a Laboratory of G Protein-Coupled Receptors, Korea University College of Medicine, 126-1 Anam-dong 5-ga, Seongbuk-gu, Seoul 136-705, Republic of Korea b Department of Life Science, Gwangju Institute of Sciences and Technology, Gwangju 500-712, Republic of Korea c INSERM U413, Laboratory of Cellular and Molecular Neuroendocrinology, European Institute for Peptide Research (IFRMP 23), University of Rouen, Mont-Saint-Aignan 76821, France d Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea 1. Introduction Kisspeptin is a neuropeptide that is involved in neuroendo- crine regulation of reproduction and tumor metastasis. Kisspeptin, the product of the KiSS-1 gene, is processed to a 54-amino acid with a C-terminal amidation. This product has been named metastin-54 or kisspeptin-54 [10,19]. Shorter C- terminally amidated peptides consisting of 13 and 14 amino acids have also been isolated from the human placenta [8].A synthetic Kiss-10 (metastin-45–54), corresponding to the C- terminal decapeptide, has been shown to be as potent as native kisspeptins [19]. KiSS-1 genes have been identified in many mammalian species including rat and mouse [27,28]. The sequence of the C-terminal decapeptide is highly peptides 30 (2009) 171–179 article info Article history: Received 27 February 2008 Received in revised form 25 April 2008 Accepted 28 April 2008 Published on line 4 May 2008 Keywords: Bullfrog GPR54 Xenopus Kisspeptin Ligand selectivity Signaling pathway abstract Kisspeptin and its receptor, GPR54, play important roles in mammalian reproduction and cancer development. However, little is known about their function in nonmammalian species. In the present study, we have isolated the cDNA encoding the kisspeptin receptor, GPR54, from the bullfrog, Rana catesbeiana. The bullfrog GPR54 (bfGPR54) cDNA encodes a 379-amino acid heptahelical G protein-coupled receptor. bfGPR54 exhibits 45–46% amino acid identity with mammalian GPR54s and 70–74% identity with fish GPR54s. RT-PCR analysis showed that bfGPR54 mRNA is highly expressed in the forebrain, hypothalamus and pituitary. Upon stimulation by synthetic human kisspeptin-10 with Phe-amide residue at the C-terminus (h-Kiss-10F), bfGPR54 induces SRE-luc activity, a PKC-specific reporter, evidencing the PKC-linked signaling pathway of bfGPR54. Using a blast search, we found a gene encoding a kisspeptin-like peptide in Xenopus. The C-terminal decapeptide of Xeno- pus kisspeptin shows higher amino acid sequence identity to fish Kiss-10s than mammalian Kiss-10s. A synthetic Xenopus kisspeptin peptide (x-Kiss-12Y) showed a higher potency than mammalian Kiss-10s in the activation of bfGPR54. This study expands our under- standing of the physiological roles and molecular evolution of kisspeptins and their receptors. # 2008 Elsevier Inc. All rights reserved. * Corresponding author. Tel.: +82 2 920 6090; fax: +82 2 921 4355. E-mail address: jyseong@korea.ac.kr (J.Y. Seong). 1 These authors contributed equally to this work. available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/peptides 0196-9781/$ – see front matter # 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2008.04.015