The 56-59-kilodalton protein identified in untransformed steroid receptor complexes is a unique protein that exists in cytosol in a complex with both the 70- and 90-kilodalton heat shock proteins

Abstract

It has previously been shown that 9S, untransformed progestin, estrogen, androgen, and glucocorticoid receptor complexes in rabbit uterine and liver cytosols contain a 59-kDa protein [Tai, P. K., Maeda, Y., Nakao, K., Wakim, N. G., Duhring, J. L., & Faber, L. E. (1986) Biochemistry 25, 5269-5275]. In this work we show that the monoclonal antibody KN 382/EC1 raised against the rabbit 59-kDa protein reacts with 9S, untransformed glucocorticoid receptor complexes in cytosol prepared from human IM-9 lymphocytes but not with 4S salt-transformed receptors. The human protein recognized by the EC1 antibody is a 56-kDa protein (p56) of moderate abundance located predominantly in the cytoplasm by indirect immunofluorescence. There are at least six isomorphs of p56 by two-dimensional gel analysis. N-Terminal sequencing (20 amino acids) shows that p56 is a unique human protein. When p56 is immunoadsorbed from IM-9 cell cytosol, both the 70- and 90-kDa heat shock proteins are coadsorbed in an immune-specific manner. Neither heat shock protein reacts directly with the EC1 antibody. We conclude that p56 exists in cytosol in a higher order complex containing hsp70 and hsp90, both of which in turn have been found to be associated with untransformed steroid receptors.

Subunit composition of the untransformed glucocorticoid receptor in the cytosol and in the cell

“…On the basis of previous observations (Tai et al, 1986;Renoir et al, 1990;Sanchez et al, 1990). we have hypothesised that p59 is the human homologue of the 59-kDa rabbit protein associated with steroid receptors.…”

“…3 indicate extensive structural similarity between glucocorticoid receptors from normal rat liver and transformed rat and human cell lines. It has been reported that only part of the molybdate-stabilized cytosolic glucocorticoid receptor from either rabbit liver (Tai et al, 1986), human IM-9 cells (Sanchez et al, 1990) or calf uterus (Renoir et al, 1990) reacted with the ECI monoclonal antibody against the 59-kDa component of the rabbit steroid receptors (Tai and Faber, 1985;Tai et al, 1986). Several lines of evidence therefore suggest that the 288-kDa complex is a distinct form of the untransformed cytosolic receptor.…”

“…4C), which is immunologically related to the 59-kDa component of rabbit steroid receptors recognized by the ECI antibody (Tai and Faber, 1985;Tai et al, 1986). Recent evidence has suggested that in the cytosol the 56-kDa protein and hsp90 form part of the untransformed glucocorticoid receptor, as well as of a complex containing hsp70 and three unidentified proteins of molecular mass 120, 35 and 23 kDa (Sanchez et al, 1990). The question then arises, whether these receptor-bound proteins can mediate formation of higher receptor oligomers.…”

“…It has been reported that the immunoadsorbed untransformed receptor from mouse thymoma cells contains 2 mol hsp90/mol steroid-binding subunit (Mendel and Orti, 1988) and that rat liver cytosolic receptor is a trimeric complex, containing two molecules of hsp90 (Denis et al, 1988;Lefebvre et al, 1989). However, there is evidence that, in addition to hsp90, distinct unidentified proteins associate with the untransformed cytosolic receptor from different sources (Tai et al, 1986;Silva and Cidlowski, 1989; Renoir et al, 1990;Sanchez et al, 1990). Based on the finding that minimally washed immunoadsorbed receptor from L cell cytosol receptor contain at least 4 mol hsp90/mol steroid-binding subunit, Bresnick et al (1990) have recently proposed that the native untransformed receptor is a large oligomer.…”

“…However, there is evidence that, in addition to hsp90, distinct unidentified proteins associate with the untransformed cytosolic receptor from different sources (Tai et al, 1986;Silva and Cidlowski, 1989;Renoir et al, 1990;Sanchez et al, 1990). Based on the finding that minimally washed immunoadsorbed receptor from L cell cytosol receptor contain at least 4 mol hsp90/mol steroid-binding subunit, Bresnick et al (1990) have recently proposed that the native untransformed receptor is a large oligomer.…”