Several nuclear proteins, including steroid hormone receptors, have been shown to shuttle continuously between the nucleus and the cytoplasm. The mechanism of entry of proteins into the nucleus is well documented, whereas the mechanism of their outward movement into the cytoplasm is not understood. We have grafted the nuclear localization signals of the progesterone receptor or the simian virus 40 large tumor antigen onto beta-galactosidase. These additions were shown to impart to the protein the ability to shuttle between the nucleus and the cytoplasm. Microinjected proteins devoid of a nuclear localization signal were unable to exit from the nucleus. The same nuclear localization signals are thus involved in both the inward and the outward movement of proteins through the nuclear membrane. We also show that although the nuclear import requires energy, the nuclear export does not. These results suggest that the nucleocytoplasmic shuttling may be a general phenomenon for nuclear proteins that could possibly undergo modifications in the cytoplasm and exert some biological activities there. These conclusions also imply that at least part of the cellular machinery involved in the nuclear import of proteins may function bidirectionally.
Previous studies on glucocorticoid receptors have suggested the existence of interactions between the receptor and microtubule or actin networks. It was hypothesized that such interactions may contribute to the guidance of steroid hormone receptors towards the nucleus. We used a permanent L cell line expressing the delta 638-642 progesterone receptor. This mutant has all the characteristics of the wild type receptor except that the deletion of five amino acids inactivates the constitutive karyophilic signal. Consequently, the receptor is cytoplasmic in the absence of hormone but is shifted into the nucleus when administration of hormone activates the second karyophilic signal. Optical microscopy and confocal laser microscopy were used in intact cells or in cells depleted of soluble elements by permeabilization with detergents. By immunofluorescence, the receptor was found to be mainly concentrated in the perinuclear area. A small fraction of progesterone receptor (PR) persisted in this region after Triton X100 treatment. These observations suggested that the receptor could interact with some insoluble constituent(s) of the cytoplasm. However, careful colocalization studies showed that this heterogenous distribution was not due to interactions with microtubule, microfilament, or intermediate filament networks. Functional involvement of these networks in the translocation of the receptor into the nucleus was studied after cell treatment with cytoskeletal drugs such as nocodazole, demecolcine and cytochalasin. None of these compounds prevented or even delayed the hormone-dependent transfer of delta 638-642 PR into the nucleus. Similar conclusions were reached with the wild type receptor expressed by transfection in Cos-7 cells. PR was shifted from the nucleus into the cytoplasm by administration of energy-depleting drugs. After disruption of the various cytoskeletal networks normal nuclear reaccumulation of the receptor was observed when these drugs were removed. The results thus suggest that the progesterone receptor is not colocalized with the main cytoskeletal components. Disruption of the cytoskeletal networks does not prevent its nuclear translocation. Thus, karyophilic signals and interactions with the nuclear pore seem to be the primary determinants of the cellular traffic of the progesterone receptor.
The nuclear localization of the progesterone receptor is mediated by two signal sequences: one is constitutive and lies in the hinge region (between the DNA and steroid binding domains), the other is hormone-dependent and is localized in the second zinc finger of the DNA binding domain. The use of various inhibitors of energy synthesis in cells expressing permanently or transiently the wild-type receptor or a receptor mutated within the nuclear localization signals, demonstrated that the nuclear residency of the receptor reflects a dynamic situation: the receptor diffusing into the cytoplasm and being constantly and actively transported back into the nucleus. The existence of this nucleo-cytoplasmic shuttle mechanism was confirmed by receptor transfer from one nucleus to the other in heterokaryons. Preliminary evidence was obtained, using oestrogen receptor, that this phenomenon may be of general significance for steroid receptors.
