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

Previous work has identified the parathyroid hormone-related protein (PTHrP ) nucleolar targeting signal (NTS) as both necessary and sufficient for loc alization of PTHrP to the nucleus and nucleolus of a variety of cells where it is believed to participate in the regulation of cell proliferation, dif ferentiation, and apoptotic cell death. The mechanism whereby a secreted pe ptide, such as PTHrP, gains access to the nuclear compartment remains a que stion of debate. The current work examines the possibility that exogenous P THrP is internalized and transported to the nuclear compartment by a mechan ism that is dependent on preservation of the PTHrP NTS. Transiently express ed, PTHrP(1-141) was detected at the cell surface as well as in the cytopla smic and nuclear compartments of COS-1 cells. Deletion of the NTS, or mutat ion of the conserved GxKKxxK motif within the NTS, effectively prevented bo th cell-surface binding and nuclear/nucleolar accumulation of PTHrP(1-141). A biotinylated peptide corresponding to the PTHrP NTS (PTHrP-NTS-biotin) w as internalized and translocated to the nucleus and nucleolus in a time-, t emperature-, and concentration-dependent manner, whereas a peptide represen ting a similar bipartite NTS from Nucleolin was not. Internalization and nu cleolar 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 pep tide, whereas pretreatment with PTHrP(1-34) or PTHrP(67-86) was not. These observations demonstrate that binding of exogenous, full-length PTHrP to th e cell. surface is mediated through a conserved motif embedded in the NTS a nd suggest that internalization and nucleolar targeting of an NTS peptide a re mediated through binding to a cell surface protein distinct from the PTH /PTHrP receptor. In total, the data support the hypothesis that secreted PT HrP(1-141) can be endocytosed and targeted to the nucleolus through a mecha nism that is dependent on preservation of a core motif within the PTHrP NTS .