Parathyroid hormone-related protein (
PTHrP), expressed in a range of
tumors, has endocrine, autocrine/paracrine, and intracrine actions, some of which relate to its ability to localize in the nucleus. Here we show for the first time that extracellularly added human
PTHrP (
amino acids 1-108) can be taken up specifically by receptor-expressing UMR106.01
osteogenic sarcoma cells and accumulate to quite high levels in the nucleus and nucleolus within 40 min. Quantitation of recognition by the nuclear localization sequence (NLS)-binding
importin subunits indicated that in contrast to
proteins containing conventional NLSs,
PTHrP is recognized exclusively by
importin beta and not by
importin alpha. The sequence of
PTHrP responsible for binding was mapped to
amino acids 66-94, which includes an SV40 large
tumor-antigen NLS-like sequence, although sequence determinants amino-terminal to this region were also necessary for high affinity binding (apparent dissociation constant of approximately 2 nM for
importin beta). Nuclear import of
PTHrP was assessed in vitro using purified components, demonstrating that
importin beta, together with the
GTP-binding protein Ran, was able to mediate efficient nuclear accumulation in the absence of
importin alpha, whereas the addition of nuclear transport factor NTF2 reduced transport. The
polypeptide ligand PTHrP thus appears to be accumulated in the nucleus/nucleolus through a novel, NLS-dependent nuclear import pathway independent of
importin alpha and perhaps also of NTF2.