Delimitation of two regions in the 90-kDa heat shock protein (Hsp90) able to interact with the glucocorticosteroid receptor (GR)

The role of the 90-kDa heat shock protein (Hsp90) as a chaperone and its re gulatory functions for cellular proteins such as the glucocorticosteroid re ceptor (GR) depends on the direct interaction of the Hsp90 with the corresp onding protein as part of a multiprotein complex. The search for the amino acid sequence(s) in Hsp90 involved in interaction with the human GR has bee n carried out by mutational deletion analysis in whole cells, studying the effects of interaction on the nucleocytoplasmic distributions of transientl y expressed Hsp90 and GR derivatives in COS-7 cells. Using a recently devel oped confocal microscopic immunofluorescence method that allows quantificat ion of the nucleocytoplasmic ratios of the proteins in individual cells and statistical comparison of cell populations, two subregions of the Hsp90 mo lecule have been defined that allow interaction with GR (residues 206-291 a nd 446-581). The latter region may play a fundamental role in the interacti on, while the former may merely stabilize the binding to GR of the intact H sp90 molecule. Moreover, the dissection of the Hsp90 molecule allowed us to define two regions displaying nuclear localization activity (residues 1-20 6 and 381-581), followed by two regions having a predominantly cytoplasmic localization activity (residues 287-381 and 581-728) and counteracting the nuclear localization activities. (C) 1999 Academic Press.