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J. Biol. Chem., Vol. 282, Issue 36, 26354-26360, September 7, 2007

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Peptidomic Identification and Biological Validation of Neuroendocrine Regulatory Peptide-1 and -2^*

Hideki Yamaguchi¹, Kazuki Sasaki¹, Yoshinori Satomi^¶1, Takuya Shimbara, Haruaki Kageyama^||, Muhtashan S. Mondal, Koji Toshinai, Yukari Date, Luis J. González^**, Seiji Shioda^||, Toshifumi Takao^¶, Masamitsu Nakazato², and Naoto Minamino³

From the Division of Neurology, Respirology, Endocrinology, and Metabolism, Department of Internal Medicine, Miyazaki Medical College, University of Miyazaki, Kihara, Kiyotake, Miyazaki 889-1692, Japan, the Department of Pharmacology, National Cardiovascular Center Research Institute, Fujishirodai, Suita, Osaka 565-8565, Japan, ^¶Laboratory of Protein Profiling and Functional Proteomics, Institute for Protein Research, Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan, ^||Department of Anatomy, Showa University School of Medicine, Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan, and ^**Physical-Chemistry Division, Center for Genetic Engineering and Biotechnology, P. O. Box 6162, Havana, Cuba

Recent advances in peptidomics have enabled the identification of previously uncharacterized peptides. However, sequence information alone does not allow us to identify candidates for bioactive peptides. To increase an opportunity to discover bioactive peptides, we have focused on C-terminal amidation, a post-translational modification shared by many bioactive peptides. We analyzed peptides secreted from human medullary thyroid carcinoma TT cells that produce amidated peptides, and we identified two novel amidated peptides, designated neuroendocrine regulatory peptide (NERP)-1 and NERP-2. NERPs are derived from distinct regions of the neurosecretory protein that was originally identified as a product of a nerve growth factor-responsive gene in PC12 cells. Mass spectrometric analysis of the immunoprecipitate using specific antibodies as well as reversed phase-high performance liquid chromatography coupled with radioimmunoassay analysis of brain extract demonstrated the endogenous presence of NERP-1 and NERP-2 in the rat. NERPs are abundant in the paraventricular and supraoptic nuclei of the rat hypothalamus and colocalized frequently with vasopressin but rarely with oxytocin. NERPs dose-dependently suppressed vasopressin release induced by intracerebroventricular injection of hypertonic NaCl or angiotensin II in vivo. NERPs also suppressed basal and angiotensin II-induced vasopressin secretion from hypothalamic explants in vitro. Bioactivity of NERPs required C-terminal amidation. Anti-NERP IgGs canceled plasma vasopressin reduction in response to water loading, indicating that NERPs could be potent endogenous suppressors of vasopressin release. These findings suggest that NERPs are novel modulators in body fluid homeostasis.

Received for publication, February 26, 2007 , and in revised form, June 25, 2007.

^* This work was supported in part by the 21st Century COE Program of the Ministry of Education, Culture, Sports, Science, and Technology (to M. N.), Takeda Science Foundation (to H. Y.), and the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (to N. M.) of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ These authors contributed equally to this work.

² To whom correspondence may be addressed. Tel.: 81-985-85-2965; Fax: 81-985-85-1869; E-mail: nakazato{at}med.miyazaki-u.ac.jp. ³ To whom correspondence may be addressed. Tel.: 81-6-6833-5012; Fax: 81-6-6835-5349; E-mail: minamino{at}ri.ncvc.go.jp.

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