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Journal of Biochemistry Advance Access originally published online on January 22, 2008
Journal of Biochemistry 2008 143(4):569-579; doi:10.1093/jb/mvn002
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© 2008 The Japanese Biochemical Society.

Cleavage of the ER-Targeting Signal Sequence of Parathyroid Hormone-related Protein is Cell-Type-Specific and Regulated in Cis by its Nuclear Localization Signal

Yoshihiro Amaya1,*, Toshiki Nakai2, Keiichi Komaru3, Masayuki Tsuneki1 and Satoshi Miura2

1Division of Biochemistry, Niigata University Graduate School of Medical and Dental Sciences, 2-5274 Gakkocho-dori, Chuo-ku, Niigata 951-8514; 2Radioisotope Research Center, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004; and 3Kitasato Junior College of Health and Hygienic Sciences, 500 Kurotsuchishinden, Minamiuonuma, Niigata 949-7421, Japan

*To whom correspondence should be addressed. Tel: +81-25-227-2830, Fax: +81-25-227-0803, E-mail: amaya{at}dent.niigata-u.ac.jp

Received October 3, 2007; Accepted December 29, 2007


  Abstract

Prepro-parathyroid hormone-related protein (ppPTHrP) has two targeting signals, an N-terminal signal sequence and a nuclear localization signal (NLS). In fact, the protein is not only secreted from the cell but also found in the nucleus and/or nucleolus. In order to understand the function of the PTHrP signal sequence for the dual localization, the signal sequence cleavage of a series of ppPTHrP deletion mutants fused to Escherichia coli leader peptidase was analysed in vitro and in several cell lines. Efficiency of the PTHrP signal sequence cleavage was intrinsically low in the in vitro reconstitution system. In cultured cells, cleavage efficiency of the PTHrP signal sequence varied significantly, being lowest in COS-1 cells, but rising in HeLa, HEK293 and CV-1 cells. However, virtually complete signal sequence cleavage was observed in CHO cells. In addition, the NLS of PTHrP had a negative effect on its own signal sequence cleavage, which could be enhanced by deletion of the spacer sequence between the signal sequence and the NLS. There was a roughly inverse relationship between the signal sequence cleavage and the nuclear localization of PTHrP. Thus, the final destination of PTHrP could be regulated at the ER membrane.

Key Words: dual localization, endoplasmic reticulum, nuclear localization signal, parathyroid hormone-related protein, signal sequence

Abbreviations: DMEM, Dulbecco's modified Eagle's medium; ER, endoplasmic reticulum; GL, {alpha}2u-globulin; GST, glutathione-S-transferase; LP, leader peptidase; MBP, maltose binding protein; NLS, nuclear localization signal; PMSF, phenylmethylsulphonyl fluoride; PNGase, peptide: N-glycosidase; PTH, parathyroid hormone; PTHrP, parathyroid hormone-related protein; ppPTHrP, prepro-parathyroid hormone-related protein; SRP, signal recognition particle


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