Molecular dissection of the importin β1-recognized nuclear targeting signal of parathyroid hormone-related protein

Produced by various types of solid tumors, parathyroid hormone-related protein (PTHrP) is the causative agent of humoral hypercalcemia of malignancy. The similarity of PTHrP's amino-terminus to that of parathyroid hormone enables it to share some of the latter's signalling properties, but its carboxy-terminus confers distinct functions including a role in the nucleus/nucleolus in reducing apoptosis and enhancing cell proliferation. PTHrP nuclear import occurs via a novel importin β1-mediated pathway. The present study uses several different direct binding assays to map the interaction of PTHrP with importin using a series of alanine mutated PTHrP peptides and truncated human importin β1 derivatives. Our results indicate that PTHrP amino acids 83-93 (KTPGKKKKGK) are absolutely essential for importin β1 recognition with residues 71-82 (TNKVETYKEQPL) additionally required for high affinity binding; residues 380-643 of importin β1 are required for the interaction. Binding of importin β1 to PTHrP is reduced in the presence of the GTP-bound but not GDP-bound form of the guanine nucleotide binding protein Ran, consistent with the idea that RanGTP binding to importin is involved in the release of PTHrP into the nucleus following translocation across the nuclear envelope. This study represents the first detailed examination of a modular, non-arginine-rich importin β1-recognized nuclear targeting signal.