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We investigated the ubiquitously expressed hsp70-associating protein Hap46, which is also called RAP46 and is homologous to BAG-1, for activities independent of hsp70 interactions. We observed in vitro binding to various DNA fragments but detected no apparent sequence specificity. Deletion of the amino-terminal decapeptide, which contains two clusters of three basic amino acids each, abolished the DNA-binding ability of Hap46. Similarly, exchange of either of these positively charged clusters for three alanines resulted in loss of DNA binding. Using a fusion of Hap46 and green fluorescent protein, we found preferential accumulation in cell nuclei on heat stress as compared with unstressed cells. The repressive effect of heat shock on overall transcriptional activity in human DU145 carcinoma cells was largely prevented when Hap46 was overexpressed by transfection. Such overproduction of Hap46 also resulted in enhanced expression of specific reporter gene constructs and in increased levels of mRNAs specific for hsp70 and hsp40 after temperature stress. In vitro transcription with nuclear extracts was stimulated greatly by Hap46. Like DNA binding, transcriptional enhancement required amino-terminally located basic amino acid residues but not the carboxyl-terminal portion of Hap46 known to participate in hsp70 interaction. Our results show that Hap46 is a bifunctional protein that can interact with both hsp70s and DNA, employing different portions of the molecule. They also suggest that Hap46 is involved in temperature-sensitive regulation of transcription, acting as a general transcriptional activator.

In search of proteins which interact with activated steroid hormone receptors, we screened a human liver [lambda]gt11 expression library with the glucocorticoid receptor. We identified and cloned a cDNA sequence of 1322 bp that encodes a protein of 274 aa. This protein consists predominantly of hydrophilic amino acids and contains a putative bipartite nuclear localization signal. The in vitro translated receptorassociating protein runs in SDS/polyacrylamide gels with an apparent molecular mass of 46 kDa. By use of the bacterially expressed fusion protein with glutathione S-transferase we have found that interaction is not limited to the glueocorticoid receptor but included other nuclear receptors - most notably, the estrogen and thyroid receptors. Binding also occurs with the glucocorticoid receptor complexed with the antiglucocorticoid RU 38486, with the estrogen receptor complexed with the antiestrogen 4-hydroxytamoxifen or ICI 164,384, and even with receptors not complexed with ligand. Association with steroid hormone receptors depends on prior receptor activation - i.e., release from heat shock proteins. The sequence identified here appears to be a general partner protein for nuclear hormone receptors, with the gene being expressed in a variety of mammalian tissues.

Analysis of the protein subunit structure of nonactivated estrogen receptor of human MCF-7 mammary carcinoma cells shows that the receptor is a heterotetramer consisting of one receptor polypeptide associated with two hsp90 molecules and one p59 subunits. The molecular architecture of the receptor is similar to glucocorticoid and progesterone receptors.

The bigger B receptor, which was separated from the A form by immunoaffinity chromatography, formed the focus of a study of the subunit structure in the analysis of nonactivated progestrone receptors in extracts of human T47D mammary carcinoma cells. The patterns of labeled band in SDS gels, obtained by the progressive cross-linking of the photo affinity-labeled receptor, suggest a heterotetrameric structure.