Nucleocytoplasmic shuttling of the mineralocorticoid receptor (MR): Differential properties of the hsp90-based heterocomplex, the nuclear localization signal 1 (NL1) and the DNA binding domain (DBD) upon binding of natural and synthetic ligands

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

New Orleans, USA

Congreso; 86th Annual Meeting of the Endocrine Society; 2004

Endocrine Society

A heuristic and unproven notion posits that hsp90 rapidly dissociates from receptors (transformation) upon steroid binding. Studying the MR signalling pathway has led us to question this model. Untrasformed MR (9.5 S) is recovered in the nucleus up to 10 min after steroid binding. The nuclear 9.5 S isoform is shifted to 11.5 S with an anti-hsp90 IgM, and hsp90 co-immunoprecipitates with MR. Thus, transformation is not an early event. Immunoprecipitation of cytoplasmic MR also co-adsorbs the immunophilins (IMMs) FKBP51 and FKBP52, the protein-phosphatase PP5 and the motor protein complex dynein-dynactin. Upon binding of aldosterone (ALDO), FKBP52, dynein and p50/dynactin-2 are recruited to MR, whereas FKBP51 dissociates and PP5 remains unchanged. However, the binding of 11,19-oxidoprogesterone (OP) recruits less FKBP52 and dynein, and more PP5. Some FKBP51 still remains in the complex. As a consequence, the nuclear translocation rate of OP/MR is slower (t0.5=15 min) than that measured for ALDO/MR (t0.5=5 min).When digitonin-permeabilized cells are incubated with extracts of 9.5 S MR, both ALDO and OP translocate MR to the nucleus in a process inhibited by NL1 peptide. Nonetheless, DEPC-transformed MR is only 50% nuclear, suggesting that the sole exposure of NL1 in the absence of ligand is insufficient to fully activate MR. In intact cells, ALDO/MR localizes in nuclear loci and OP/MR shows a diffuse pattern. However, both steroids induce equal maximum transcriptional activity (25-fold). Suboptimal induction by ALDO (5-fold) is potentiated (80-fold) by inactive concentrations of OP, which suggests an allosteric interaction. This potentiation is increased by overexpression of p300. Limited proteolysis of ALDO/MR or OP/MR yields different fragments, which explains all previous results: the ligands promote different conformational states of MR. In permeabilized cells, steroid withdrawal recycles MR to the cytoplasm in an ATP- and molybdate-dependent manner only. The nuclear export of ALDO/MR (but not OP/MR) is abrogated by the DBD peptide that separates the two Zn-fingers of MR, suggesting that the DBD can affect nuclear export, perhaps by masking binding to calreticulin. In conclusion, the retrograde movement of MR requires hsp90-FKBP52, and is powered by dynein, so MR’s transformation may be nuclear rather than cytoplasmic. The ligand plays a key role in recruiting IMMs, exposing the NL1, regulating transcriptional activity and retaining MR in the nucleus