Discrimination between NL1- and NL2-mediated nuclear localization of the glucocorticoid receptor

Glucocorticoid receptor (GR) cycles between a free liganded form that is lo calized to the nucleus and a heat shock protein (hsp)-immunophilin-complexe d, unliganded form that is usually localized to the cytoplasm but that can also be nuclear. In addition, rapid nucleocytoplasmic exchange or shuttling of the receptor underlies its localization. Nuclear import of liganded GR is mediated through a well-characterized sequence, NL1, adjacent to the rec eptor DNA binding domain and a second, uncharacterized motif, NL2, that ove rlaps with the ligand binding domain. In this study we report that rapid nu clear import (half-life [t(1/2)] of 4 to 6 min) of agonist- and antagonist- treated GR and the localization of unliganded, hsp-associated GRs to the nu cleus in G(0) are mediated through NL1 and correlate with the binding of GR to pendulin/importin alpha. By contrast, NL2-mediated nuclear transfer of GR occurred more slowly (t(1/2) = 45 min to 1 h), was agonist specific, and appeared to be independent of binding to importin alpha. Together, these r esults suggest that NL2 mediates the nuclear import of GR through an altern ative nuclear import pathway. Nuclear export of GR was inhibited by leptomy cin B, suggesting that the transfer of GR to the cytoplasm is mediated thro ugh the CRM1-dependent pathway. Inhibition of GR nuclear export by leptomyc in B enhanced the nuclear localization of both unliganded, wild-type GR and hormone-treated NL1(-) GR These results highlight that the subcellular loc alization of both liganded and unliganded GRs is determined, at least in pa rt, by a flexible equilibrium between the rates of nuclear import and expor t.