The nucleolar targeting signal (NTS) of parathyroid hormone related protein mediates endocytosis and nucleolar translocation

J Bone Miner Res. 1999 Sep;14(9):1493-503. doi: 10.1359/jbmr.1999.14.9.1493.

Abstract

Previous work has identified the parathyroid hormone-related protein (PTHrP) nucleolar targeting signal (NTS) as both necessary and sufficient for localization of PTHrP to the nucleus and nucleolus of a variety of cells where it is believed to participate in the regulation of cell proliferation, differentiation, and apoptotic cell death. The mechanism whereby a secreted peptide, such as PTHrP, gains access to the nuclear compartment remains a question of debate. The current work examines the possibility that exogenous PTHrP is internalized and transported to the nuclear compartment by a mechanism that is dependent on preservation of the PTHrP NTS. Transiently expressed, PTHrP(1-141) was detected at the cell surface as well as in the cytoplasmic and nuclear compartments of COS-1 cells. Deletion of the NTS, or mutation of the conserved GxKKxxK motif within the NTS, effectively prevented both cell-surface binding and nuclear/nucleolar accumulation of PTHrP(1-141). A biotinylated peptide corresponding to the PTHrP NTS (PTHrP-NTS-biotin) was internalized and translocated to the nucleus and nucleolus in a time-, temperature-, and concentration-dependent manner, whereas a peptide representing a similar bipartite NTS from Nucleolin was not. Internalization and nucleolar targeting of PTHrP-NTS-biotin were indistinguishable in CFK2 cells, which express the common PTH/PTHrP receptor, and in 27m21 cells, which do not. In addition, pretreatment with a saturating dose of synthetic PTHrP(74-113) was capable of abrogating nucleolar accumulation of the PTHrP-NTS peptide, whereas pretreatment with PTHrP(1-34) or PTHrP(67-86) was not. These observations demonstrate that binding of exogenous, full-length PTHrP to the cell surface is mediated through a conserved motif embedded in the NTS and suggest that internalization and nucleolar targeting of an NTS peptide are mediated through binding to a cell surface protein distinct from the PTH/PTHrP receptor. In total, the data support the hypothesis that secreted PTHrP(1-141) can be endocytosed and targeted to the nucleolus through a mechanism that is dependent on preservation of a core motif within the PTHrP NTS.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding, Competitive
  • Biological Transport
  • COS Cells
  • Cell Nucleolus / metabolism*
  • Endocytosis*
  • Molecular Sequence Data
  • Parathyroid Hormone / metabolism*
  • Parathyroid Hormone-Related Protein
  • Protein Sorting Signals / metabolism*
  • Proteins / metabolism*
  • Receptor, Parathyroid Hormone, Type 1
  • Receptors, Parathyroid Hormone / metabolism
  • Signal Transduction*

Substances

  • Parathyroid Hormone
  • Parathyroid Hormone-Related Protein
  • Protein Sorting Signals
  • Proteins
  • Receptor, Parathyroid Hormone, Type 1
  • Receptors, Parathyroid Hormone