Importin β recognizes parathyroid hormone-related protein with high affinity and mediates its nuclear import in the absence of importin α

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 β and not by importin α. 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 ~2 nM for importin β). Nuclear import of PTHrP was assessed in vitro using purified components, demonstrating that importin β, together with the GTP-binding protein Ran, was able to mediate efficient nuclear accumulation in the absence of importin α, 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 α and perhaps also of NTF2.