IN-VIVO FUNCTIONAL PROTEIN-PROTEIN INTERACTION - NUCLEAR TARGETED HSP90 SHIFTS CYTOPLASMIC STEROID-RECEPTOR MUTANTS INTO THE NUCLEUS

In target tissue extracts, heat shock protein hsp90 has been found ass ociated to all unliganded steroid receptors. Modulation of important f unctions of these receptors, including prevention of DNA binding and o ptimization of transcriptional activity, has been attributed to hsp90. However no unequivocal in vivo demonstration of interaction between r eceptors and hsp90 has been presented. We targeted chicken hsp90, a ma inly cytoplasmic protein, with the nucleoplasmin nuclear localization signal (90NLS). After transfection into COS-7 cells, 90NLS was found i n the nucleus with specific immunofluorescence and confocal microscopy techniques. A human glucocorticosteroid receptor mutant devoid of NLS sequence was also expressed in COS-7 cells and found exclusively cyto plasmic. Coexpression of 90NLS and of the cytoplasmic human glucocorti costeroid receptor mutant led to complete nuclear localization of the receptor, indicating its piggyback transport by 90NLS and thus physica l and functional interaction between the two proteins in the absence o f hormone. The same nuclear localization was obtained after cotransfec tion of 90NLS and a cytoplasmic rabbit progesterone receptor mutant. F inally, coexpression of wild-type rabbit progesterone receptor (nuclea r) and wild-type hsp90 (cytoplasmic) into COS-7 cells provoked partial relocalization of hsp90 into the nucleus. These experiments lay the g roundwork on which to study hsp90 as a chaperone, regulating activitie s of steroid receptors and possibly participating in their nuclear-cyt oplasmic shuttling.