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Accumulated data suggest that endothelial cells express specific receptors for several peptide and (glyco)protein hormones that may transport hormones across the cell to be delivered to the interstitial fluid and tissue target cells. Surprisingly, very little information is available on the actual endothelial organelles involved in this cellular process. In the present study the transfer of follicle-stimulating hormone (FSH) through the endothelial barrier of rat testes was examined by analysing the binding and transport of gold-tagged recombinant human (rh)FSH under various conditions using electron microscopy. At 4 degrees C the probe bound specifically to the luminal surface of the endothelial cells without internalization. The use of 125I-rhFSH, which allows precise quantitation of the binding, confirmed the specificity of hormone interaction with the testicular microvasculature. At 37 degrees C the hormone was internalized via coated pits and vesicles into an extensive subluminal tubulo-vesicular compartment and was transported across the endothelium via a system of tubules and vesicles. Moreover, monoclonal antibodies against the FSH receptor ectodomain coupled to colloidal gold followed the same route. In contrast, a non specific, fluid-phase uptake via caveolae was observed for a major plasma protein - rat serum albumin and a fluid-phase tracer - peroxidase. These results suggest that FSH transcytosis across the testicular endothelial barrier is receptor-mediated and involves luminal uptake via coated pits/vesicles, sorting at the level of luminal early endosomes, and transcellular transport through transcytotic tubulo-vesicular organelles. Similar receptor-mediated pathways are likely to be involved in the physiological functioning of a number of other protein and peptide hormones that must translocate specifically from blood to the target cells.

Steroid hormone receptors are, in most cases, mainly nuclear proteins that undergo a continuous nucleocytoplasmic shuttling. The mechanism of the nuclear export of these proteins remains largely unknown. To approach this problem experimentally in vivo, we have prepared cell lines permanently coexpressing the wild-type nuclear progesterone receptor (PR) and a cytoplasmic receptor mutant deleted of its nuclear localization signal (NLS) [(deltaNLS)PR]. Each receptor species was deleted from the epitope recognized by a specific monoclonal antibody, thus allowing separated observation of the two receptor forms in the same cells. Administration of hormone provoked formation of heterodimers during nucleocytoplasmic shuttling and import of (deltaNLS)PR into the nucleus. Washing out of the hormone allowed us to follow the export of (deltaNLS)PR into the cytoplasm. Microinjection of BSA coupled to a NLS inhibited the export of (deltaNLS)PR. On the contrary, microinjection of BSA coupled to a nuclear export signal (NES) was without effect. Moreover, leptomycin B, which inhibits NES-mediated export, was also without effect. tsBN2 cells contain a thermosensitive RCC1 protein (Ran GTP exchange protein). At the nonpermissive temperature, the nuclear export of (deltaNLS)PR could be observed, whereas the export of NES-BSA was suppressed. Microinjection of GTPgammaS confirmed that the export of (deltaNLS)PR was not dependent on GTP hydrolysis. These experiments show that the nuclear export of PR is not NES mediated but probably involves the NLS. It does not involve Ran GTP, and it is not dependent on the hydrolysis of GTP. The nucleocytoplasmic shuttling of steroid hormone receptors thus appears to utilize mechanisms different from those previously described for some viral, regulatory, and heterogeneous ribonuclear proteins.

The signal responsible for the nuclear localization of the progesterone receptor has been characterized. It is a complex signal. The study of the mechanism of this nuclear localization has revealed that the receptor continuously shuttles between nucleus and the cytoplasm. The receptor diffuses into the cytoplasm and is constantly and actively transported back into the nucleus. The same phenomenon exists for estradiol and glucocorticoid receptors. The mechanism of entry of proteins into the nucleus is well documented, whereas the mechanism of their outward movement to the cytoplasm is not understood. We have grafted different nuclear localization signals (NLSs) onto beta-galactosidase and have studied the traffic of this protein using heterokaryons and microinjection experiments. We have demonstrated that the same NLSs are involved in both the inward and the outward movement of proteins through the nuclear membrane. 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. Using these techniques, we have shown that the two major antiprogestins, RU486 and ZK98299, act at the same distal level of hormone action.

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.

The progesterone receptor displays the typical three-domains structure of the steroid-thyroid receptor family. The central domain contains two 'zinc finger' structures responsible for the specific recognition of the cognate DNA sequences. The carboxy-terminal domain contains the hormone and anti-hormone binding site. Progesterone and synthetic progestins (R5020, Org 2058) activate the receptor, provoke its phosphorylation and DNA-binding ability and induce its regulatory activities. The antagonist RU38486 elicits the same sequence of events but leads to an abortive conclusion without specific gene transactivation. The progesterone receptor is down-regulated by its own ligand at the transcriptional level through inhibition of oestrogen receptor-mediated induction through protein-protein interactions. This mechanism is also inhibited by RU38486.

The signal responsible for the nuclear localization of the progesterone receptor has been characterized. The study of the mechanism of this nuclear localization has revealed that the receptor continuously shuttles between the nucleus and the cytoplasm. The receptor diffuses into the cytoplasm and is constantly and actively transported back into the nucleus. Preliminary evidence suggests that the same mechanism exists for estradiol and glucocorticoid receptors. Experiments designed to study the traffic of steroid hormone receptors have been applied to the determination of the molecular mechanism of action of antisteroids. Using these techniques, we have shown that two major antiprogestins, RU486 and ZK98299, act at the same point in the cell as the hormone.

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.

Deletion mutants of the rabbit progesterone receptor were used to identify two major mechanisms of its nuclear localization. A putative signal sequence, homologous to that of the SV40 large T antigen, was localized around amino acids 638-642 and was shown to be constitutively active. When amino acids 638-642 were deleted, the receptor became cytoplasmic but could be shifted into the nucleus by the addition of hormone (or anti-hormone), it was almost fully active. A second putative nuclear localization signal is located in the DNA binding domain activated either through ligand binding or through production of constitutive receptor. By deleting epitopes recognized by monoclonal antibodies, it was possible to follow different receptor mutants inside the same cells. In the absence of ligand the receptor was transferred into the nucleus as a monomer. After administration of hormone (or anti-hormone) a "cytoplasmic" monomer was transferred into the nucleus through interaction with a "nuclear" monomer. These interactions occurred through the steroid binding domains of both monomers.

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.

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.

During the past years there has been an improvement in our understanding of the molecular mechanism of action of the progesterone receptor (PR). This was due to the obtention of monoclonal antibodies against PR which allowed the first structural analyses and led to the cloning of the genes.

Deletion mutants of the rabbit progesterone receptor were used to identify two major mechanisms of its nuclear localization. A putative signal sequence, homologous to that of the SV40 large T antigen, was localized around amino acids 638-642 and shown to be constitutively active. When amino acids 638-642 were deleted, the receptor became cytoplasmic but could be shifted into the nucleus by the addition of hormone (or anti-hormone); it was almost fully active. The second mechanism consisted of the activation of the DNA binding domain. By deleting epitopes recognized by monoclonal antibodies, it was possible to follow different receptor mutants inside the same cells. In the absence of ligand, the receptor was transferred into the nucleus as a monomer. After administration of hormone (or anti-hormone) a "cytoplasmic" monomer was transferred into the nucleus through interaction with a "nuclear" monomer. These interactions occurred through the steroid binding domains of both monomers.