Dynamics of leptomycin B-sensitive nucleocytoplasmic flux of parathyroid hormone-related protein

Parathyroid hormone-related protein is responsible for hypercalcemia induce d by various tumors. The similarity of its N-terminus to that of parathyroi d hormone enables parathyroid hormone-related protein to share parathyroid hormone's signaling properties, but the rest of the molecule possesses dist inct functions including a role in the nucleus/nucleolus in reducing apopto sis and enhancing cell proliferation. We have previously shown that parathy roid hormone-related protein nuclear import is mediated by importin beta1. Here we use fluorescence recovery after photobleaching for the first time t o show that, in living cells, parathyroid hormone-related protein is export ed from the nucleus in a leptomycin B-sensitive manner, implicating CRM1 as the parathyroid hormone-related protein nuclear export receptor. Leptomyci n B treatment significantly reduced the rate of nuclear export 4-10-fold, t hereby increasing parathyroid hormone-related protein concentration in the nucleus/nucleolus about 2-fold. Intriguingly, this also led to a 2-fold red uced nuclear import rate. Inhibiting the nuclear export of a protein able t o shuttle between nucleus and cytoplasm through distinct receptors thus can also affect nuclear import, indicating that the subcellular localization o f a protein containing distinct nuclear import and export signals is the pr oduct of an integrated system. Although there have been several recent stud ies examining the dynamics of intranuclear transport using fluorescence rec overy after photobleaching, this represents, to our knowledge, the first us e of the technique to examine the kinetics of nucleocytoplasmic flux in liv ing cells.