Molecular dissection of the importin beta 1-recognized nuclear targeting signal of parathyroid hormone-related protein

Produced by various types of solid tumors, parathyroid hormone-related prot ein (PTHrP) is the causative agent of humoral hypercalcemia of malignancy. The similarity of PTHrP's amino-terminus to that of parathyroid hormone ena bles it to share some of the latter's signalling properties, but its carbox y-terminus confers distinct functions including a role in the nucleus/nucle olus in reducing apoptosis and enhancing cell proliferation. PTHrP nuclear import occurs via a novel importin beta1-mediated pathway. The present stud y uses several different direct binding assays to map the interaction of PT HrP with importin beta using a series of alanine mutated PTHrP peptides and truncated human importin beta1 derivatives. (Pur results indicate that PTH rP amino acids 83-93 (KTPGKKKKG K) are absolutely essential for importin be ta1 recognition with residues 71-82 (TNKVETYKEQPL) additionally required fo r high affinity binding; residues 380-643 of importin beta1 are required fo r the interaction. Binding of importin beta1 to PTHrP is reduced in the pre sence of the GTP-bound but not GDP-bound form of the guanine nucleotide bin ding protein Ran, consistent with the idea that RanGTP binding to importin beta is involved in the release of PTHrP into the nucleus following translo cation across the nuclear envelope. This study represents the first detaile d examination of a modular, non-arginine-rich importin beta1-recognized nuc lear targeting signal. (C) 2001 Academic Press.