title: The nucleolar targeting signal (NTS) of parathyroid hormone related protein mediates endocytosis and nucleolar translocation.
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.
authors.affiliation: Department of Medicine, McGill University, Lady Davis Institute and SMBD-Jewish General Hospital, Montréal, Canada.
authors.forename: M M
authors.initials: MM
authors.lastname: Aarts
authors.forename: A
authors.initials: A
authors.lastname: Rix
authors.forename: J
authors.initials: J
authors.lastname: Guo
authors.forename: R
authors.initials: R
authors.lastname: Bringhurst
authors.forename: J E
authors.initials: JE
authors.lastname: Henderson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D001692:Biological Transport; D019556:COS Cells; D002466:Cell Nucleolus / Q000378:metabolism*; D004705:Endocytosis; D008969:Molecular Sequence Data; D010281:Parathyroid Hormone / Q000378:metabolism*; D044162:Parathyroid Hormone-Related Protein; D021382:Protein Sorting Signals / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D021382:Protein Sorting Signals; D011506:Proteins; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone