Nucleocytoplasmic shuttling of the MR is differentially regulated by ligand binding

MARIO D. GALIGNIANA; ALBERTO A. GHINI; CARLOS P. LANTOS; GRACIELA PIWIEN PILIPUK

New Orleans, USA

Congreso; 30th International Aldosterone Conference; 2004

International Aldosterone Organization

The MR exists as a large heterocomplex composed by hsp90, hsp70, p23, and a TPR (tetratricopeptide repeat) protein such as FKBP52, FKBP51, CyP40, PP5 or hop/p60. A heuristic and unproven notion posits that, upon steroid binding, the heterocomplex rapidly dissociates from the MR (transformation). This would expose its buried nuclear localization signal (NLS), which favors a rapid translocation (t0.5= 5 min) to the nuclear compartment. Studying the translocation mechanism of corticosteroid receptors has led us to question the traditional concept that transformation is an early event. Thus, untrasformed MR (9.5 S) is recovered in the nucleus 10 min after steroid binding, a time when the MR is entirely nuclear. The nuclear 9.5 S form of MR is shifted to 11.5 S by preincubating the samples with an anti-hsp90 IgM. In addition, hsp90 is recovered with the MR or the GR as soon as these receptors become nuclear (¡Ü 15 min). Therefore, receptor transformation is not an early event. In agreement with this concept, we have reported that cytoplasmic MR associates with the motor protein dynein (Dyn) via the hsp90-binding immunophilin (IMM), FKBP52. Inasmuch as the heterocomplex is dynamic rather than static, FKBP52 can be replaced by other TPR proteins, i.e. FKBP51 or the IMM-like Ser/Thr-phosphatase, PP5. Importantly, PP5 binds Dyn as efficiently as FKBP52, and all the MR-hsp90-IMM-Dyn complexes are bound to dynactin, a protein complex required for the biological function of the motor protein. Upon binding of aldosterone (ALDO), large amounts of FKBP52, Dyn and p50/dynactin-2 are recruited to the MR; FKBP51 dissociates (presumably replaced by FKBP52) and PP5 remains almost unchanged. In contrast, the binding of the synthetic agonist 11-OP recruits more PP5 and some FKBP51 still remains bound to the heterocomplex. FKBP52 and the Dyn-dynactin complex are also recruited by 11-OP/MR, but at a lower extent than ALDO/MR complexes. As a consequence, the nuclear translocation rate of 11-OP/MR is 3-fold slower than that measured for ALDO/MR. When digitonin-permeabilized cells are incubated with extracts of 9.5 S MR, both ALDO and 11-OP translocate MR to the nucleus in a process inhibited by the NLS-1 (NL1) peptide of the SV40-large T antigen. Nonetheless, diethylpyrocarbamate-transformed MR is only 50% nuclear, which suggests that the sole exposure of the NL1 in the absence of ligand is not sufficient to fully activate the receptor. In intact cells, the nuclear ALDO/MR complex localizes in specific loci, whereas the OP/MR complex shows a more diffuse pattern. However, both steroids induce equal maximum reporter-gene activity (25-fold). Similar to the Na+-retaining effect described in vivo, a suboptimal induction by ALDO (5-fold) is potentiated (80-fold) by inactive concentrations of 11-OP. Because this potentiation is even greater if p300 is overexpressed, the recruitment of co-activators may be limiting for this allosteric mechanism. In permeabilized cells, steroid withdrawal recycles MR to the cytoplasm in an ATP- and molybdate-dependent manner. Surprisingly, the nuclear export of ALDO/MR (but not the export of 11-OP/MR) is abrogated by a DNA-binding domain (DBD) peptide that separates the two Zn-fingers of MR, suggesting that the DBD affects nuclear export. We speculate that the DBD peptide masks the binding of MR to calreticulin. Limited proteolysis of ALDO/MR or 11-OP/MR yields different degradation fragments, which may explain most of the observations reported here: both ligands promote different conformational states of MR. Consequently, this leads to the selective turnover of trafficking proteins and influences MR-dependent transcriptional activity, perhaps by a differential recruitment of co-factors. In conclusion, the retrograde movement of MR does require the binding of the hsp90-IMM complex to link the heterocomplex with Dyn motor proteins. Therefore, receptor transformation may be nuclear rather than cytoplasmic. Importantly, ligand binding to MR plays a key role in recruiting IMMs, properly exposing the NL1, regulating transcriptional activity, and retaining MR in the nuclear compartment, so it may be stated that both ligand and receptor behave as a unique functional unit