Neuropeptide Receptor Transcriptome Reveals Unidentified Neuroendocrine Pathways Naoki Yamanaka1¤, Sachie Yamamoto1, Dusˇ an Zˇ itnˇ an 2 , Ken Watanabe1, Tsuyoshi Kawada3, Honoo Satake3, Yu Kaneko 4 , Kiyoshi Hiruma 4 , Yoshiaki Tanaka5, Tetsuro Shinoda5*, Hiroshi Kataoka1* 1 Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, Japan, 2 Institute of Zoology, Slovak Academy of Sciences, Bratislava, Slovakia, 3 Suntory Institute for Bioorganic Research, Osaka, Japan, 4 Faculty of Agriculture and Life Sciences, Hirosaki University, Aomori, Japan, 5 National Institute of Agrobiological Sciences, Ibaraki, Japan Abstract Neuropeptides are an important class of molecules involved in diverse aspects of metazoan development and homeostasis. Insects are ideal model systems to investigate neuropeptide functions, and the major focus of insect neuropeptide research in the last decade has been on the identification of their receptors. Despite these vigorous efforts, receptors for some key neuropeptides in insect development such as prothoracicotropic hormone, eclosion hormone and allatotropin (AT), remain undefined. In this paper, we report the comprehensive cloning of neuropeptide G protein-coupled receptors from the silkworm, Bombyx mori, and systematic analyses of their expression. Based on the expression patterns of orphan receptors, we identified the long-sought receptor for AT, which is thought to stimulate juvenile hormone biosynthesis in the corpora allata (CA). Surprisingly, however, the AT receptor was not highly expressed in the CA, but instead was predominantly transcribed in the corpora cardiaca (CC), an organ adjacent to the CA. Indeed, by using a reverse-physiological approach, we purified and characterized novel allatoregulatory peptides produced in AT receptor-expressing CC cells, which may indirectly mediate AT activity on the CA. All of the above findings confirm the effectiveness of a systematic analysis of the receptor transcriptome, not only in characterizing orphan receptors, but also in identifying novel players and hidden mechanisms in important biological processes. This work illustrates how using a combinatorial approach employing bioinformatic, molecular, biochemical and physiological methods can help solve recalcitrant problems in neuropeptide research. Citation: Yamanaka N, Yamamoto S, Zˇ it nˇan D, Watanabe K, Kawada T, et al. (2008) Neuropeptide Receptor Transcriptome Reveals Unidentified Neuroendocrine Pathways. PLoS ONE 3(8): e3048. doi:10.1371/journal.pone.0003048 Editor: Vladimir B. Bajic, University of the Western Cape, South Africa Received May 12, 2008; Accepted August 2, 2008; Published August 25, 2008 Copyright: 2008 Yamanaka et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: N.Y. was a Research Fellow of the Japan Society for the Promotion of Science. This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN), grants from Research for the Future Program of Japan Society for the Promotion of Science (JSPS), the National Institute of Health, USA (GM 67310), and Slovak grant agencies, Agentu´ ra na podporu vy´skumu a vy´voja (APVV-51-039105) and Vedecka´ grantova´ agentu´ ra (VEGA 2-6090-26). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: shinoda@affrc.go.jp (TS); kataoka@k.u-tokyo.ac.jp (HK) € Current address: Department of Genetics, Cell Biology and Development, Howard Hughes Medical Institute, University of Minnesota, Minneapolis, Minnesota, United States of America Introduction Diverse biological processes in metazoans such as development, reproduction and behavior are regulated by neuropeptides. One of the most effective approaches for analyzing their physiological functions is to identify their receptors, most of which belong to the G protein-coupled receptor (GPCR) superfamily. In insects, Drosophila melanogaster has been used as a model organism for such an approach. To date, some forty GPCRs are categorized as neuropeptide GPCRs in Drosophila, and about two-thirds of these receptors have been ‘‘deorphanized’’ (i.e. their endogenous ligands have been found) [1,2]. In spite of this success in flies, receptors for several key neuropeptides in insect development are yet to be identified. Among such neuropeptides is allatotropin (AT), which is the only neuropeptide widely thought to stimulate juvenile hormone (JH) biosynthesis [3,4]. JH is involved in various aspects of insect physiology such as development, reproduction and polyphenism [5–7], but the regulatory mechanisms that control its biosynthesis are not well understood in part due to the lack of information on several key molecules such as the AT receptor. The identities of receptors for prothoracicotropic hormone and eclosion hormone, two other important players in the regulation of insect develop- ment, also remain uncharacterized. Considering that the target organs of these key neuropeptides are known, comprehensive tissue expression analyses of the remaining orphan receptors seems likely to be informative in identifying their receptors. With just a few exceptions, however [8,9], such spatial expression analyses have been hampered due to the small size of the Drosophila. In contrast, larger lepidopteran insects such as Manduca sexta and Bombyx mori allowed detailed tissue expression analyses of several neuropeptide GPCRs, which have helped elucidate novel functions of their ligands [10–12]. Now that a draft sequence of the Bombyx genome has been published [13,14], a comprehensive analysis of an entire neuropeptide GPCR transcriptome is feasible for the first time among lepidopterans. Using Bombyx, we report here the first comprehensive cloning and thorough analyses of neuropeptide GPCRs conducted in any organism. This approach, combined with biochemical and PLoS ONE | www.plosone.org 1 August 2008 | Volume 3 | Issue 8 | e3048