Nuclear targeting of parathyroid hormone-related protein and apoptosis

Parathyroid Hormone-related Protein (PTHrP) was first identified as a tumor-derived product responsible for Malignancy Associated Hypercalcemia. The protein was characterized based on its ability to mimic Parathyroid Hormone (PTH) in kidney and bone. PTHrP is a secretory peptide with limited homology to PTH at the amino-terminus that allows it to bind to the common PTH/PTHrP receptor (PTH1R). Normal secretion of PTHrP has now been demonstrated in a number of tissues where it acts as an autocrine/paracrine mediator of cell growth and differentiation. Much of the documented biological activity of PTHrP is mediated through PTH1R however a body of evidence indicates that PTHrP uses multiple signal pathways in target tissues. One of these pathways consists of an intracellular signal mechanism that is dependent on a bipartite nucleolar targeting signal (NTS) located between residues 87–107 of the mature protein. This sequence is similar to the NTS motifs found in the nucleolar HIV-1 proteins Tat and Rev. Studies have shown that the NTS of PTHrP is both necessary and sufficient to target PTHrP to the nucleus and nucleolus of a variety of cells. Nuclear localization has been associated with altered gene expression, proliferation and inhibition of apoptosis. The mechanism used by this secretory protein to gain access to the nucleus is largely unknown. We examined the possibility that PTHrP is internalized from the cell surface and transported to the nucleolus in a manner dependent on the NTS. Using transiently transfected COS-1 cells we demonstrated NTS dependent localization of full-length PTHrP at the plasma membrane and in nuclear compartments. Site-directed mutagenesis identified an 87GxKKxxK93 motif at the N-terminus of the NTS that is critical for nuclear/nucleolar targeting. A biotin-labeled PTHrP-NTS peptide was internalized and localized to the nucleolus in a time and dose-dependent manner whereas a similarly basic NLS peptide was not. Internalization of PTHrP-NTS was temperature dependent suggesting active endocytosis of the protein. Uptake and nucleolar localization of PTHrP-NTS occurred in both CKF2 chondrocytes that express PTH1R and 27m21 chondrocytes that do not. PTHrP-NTS internalization could be inhibited by pre-treatment of the chondrocytes with unlabeled peptides containing the PTHrP NTS but not with PTHrP-(1–34) or (67–86) peptides. Thus our findings demonstrate that the PTHrP NTS can mediate cell surface binding, internalization and nuclear/nucleolar localization via binding to a cell surface protein distinct from PTH1R. These results support our hypothesis that secreted PTHrP can translocate to the nucleolus after endocytosis from the cell surface.; We investigated whether binding to resident RNA structures targets PTHrP to the nucleolus. We have shown that transfected, in vitro translated and endogenous PTHrP bind to homopolymeric and cellular RNA with high affinity and specificity. Binding was dependent on the NTS and in particular the 87GxKKxxK93 motif. This motif is also a conserved sequence in double-stranded RNA binding proteins. This work represents the first demonstration of RNA binding by a secretory protein and predicts a role for PTHrP in regulating ribosome biogenesis in the nucleolus. (Abstract shortened by UMI.)...