Effect of Initial Subcellular Localization of Progesterone Receptor on Import Kinetics and Transcriptional Activity

Abstract

Progesterone receptors (PR) are ligand-activated transcription factors that modulate transcription by activating genes. There are two isoforms of PR, PRA and PRB. In most cell contexts, the PRA isoform is a repressor of the PRB isoform. Without hormone induction, PRA is mostly located in the nucleus whereas PRB distributes both in the nucleus and in the cytoplasm. In this paper, a new model system has been used to study the impact of initial subcellular localization, and import rate of progesterone receptor on transcriptional activity. This new model system involves using a mutant version of PRB which is found only in the cytoplasmic compartment of cells in the unliganded state, making the distribution of the receptor more homogeneous to start with compared with the previous model, wild type (wt) PRB, which has a more heterogeneous distribution (nuclear and cytoplasmic even without ligand). Import kinetics has been shown to be one of the major means by which to regulate PR transcriptional activity. Fluorescence microscopy was used to measure green fluorescent protein tagged PRB import rate into the nucleus. Luciferase reporter gene assay was used to measure transcriptional activity of PRB. In addition, a two-hybrid assay was performed to measure the interaction between PRB and importin alpha. Mutant versions of PRA and PRB with the constitutively active nuclear localization signal removed were created (PRA-NLSc mutant and PRB-NLSc mutant). These PR mutants were found to localize mainly in the cytoplasm in the absence of hormone. With addition of hormone, PR mutants translocated to the nucleus, although at a slower rate compared to wt PRB. Our results show that the activation of reporter gene transcription is proportional to the nuclear import rate of PRB-NLSc mutant, and the difference in import kinetics between wt PRB and the PRB-NLSc mutant is due to a stronger interaction of wt PRB with importin alpha. We also show that the hormone inducible NLS in PR, NLSh, is a weak nuclear localization signal even without hormone and can act as a weak hormone dependent nuclear localization signal when combined with the ligand binding domain of PR. In addition, by changing the initial subcellular localization of PRA from the nucleus to the cytoplasm, this diminished PRA's ability to act as an inhibitor of PRB.

Role of molecular chaperones and TPR-domain proteins in the cytoplasmic transport of steroid receptors and their passage through the nuclear pore

“…Interestingly, the nuclear translocation rate of GR in that NL1-deleted mutant is identical to the rate shown in cells where the hsp90·FKBP52·motor protein complex is disrupted. A similar observation has also been reported for a PR mutant where the active NLS is absent 64. As expected, this PR mutant is cytoplasmic in a medium without steroid.…”

Estrogen and progesterone receptors: from molecular structures to clinical targets

“…In addition to both PRA/B transcriptional start sites, a single nucleotide polymorphism (SNP) called PR?331G/A creates a new TATA element, which results in additional PRB transcription [58]. Using transiently transfected PR constructs, PRA expression leads mainly to gene repression, whereas PRB correlated with gene activation [59,60]. A comparison of both isoforms showed that PRA was Nterminally truncated by 165 amino acid residues resulting in a protein size of 94 kDa compared to PRB with 116 kDa (Fig.…”

“…3) [61]. In addition, PRA is mostly located in the nucleus, whereas PRB distributes between the nucleus and cytoplasm [59]. PRA and PRB are co-expressed in many cell types where they appear to be synthesized in equal proportions.…”

Visualizing the action of steroid hormone receptors in living cells

“…In most cell contexts, the PRA isoform is a repressor of the shorter PRB isoform, and without hormone induction it is mostly located in the nucleus, whereas PRB distributes both in the nucleus and in the cytoplasm. PRB accumulates in the nucleus after progesterone binding, a process that directly correlates with PR mediated transcription [Li et al, 2005; Lim et al, 1999]. …”