Evidence that heat shock protein-70 associated with progesterone receptors is not involved in receptor-DNA binding.
In the absence of hormone, human progesterone receptors (PR) are recovered in the cytosolic fraction of cell lysates as a multimeric complex containing the steroid-binding polypeptide, heat shock protein-90 (hsp90), and heat shock protein-70 (hsp70). Activated forms of human PR that acquire the ability to bind to DNA are dissociated from hsp90, but retain association with hsp70. The present study has examined whether associated hsp70 has a function in receptor-DNA binding. When activated PR was bound to specific target DNA in a gel shift assay, no hsp70 was detectable in the PR-DNA complex, as evidenced by the failure of several antibodies to hsp70 to affect the mobility or the amount of complexes. To determine whether hsp70 might indirectly influence DNA-binding activity, we have examined the effect of hsp70 dissociation on PR-DNA-binding activity. Dissociation was achieved either by treatment of immunoaffinity-purified immobilized PR complexes with ATP or by the binding of PR complexes to ATP-agarose, followed by elution with high salt. Under both conditions, dissociation from hsp70 neither enhanced nor impaired the ability of PR to bind to specific DNA. These results suggest that hsp70 is not involved in PR binding to DNA, either directly by participating in DNA binding or indirectly by modulating PR-DNA-binding activity. This implies that hsp70 functions at an earlier stage in the receptor activation pathway. Consistent with the known involvement of hsp70 in stabilizing unfolded states of other target proteins, we propose that hsp70 may assist in nuclear transport of PR or in assembly-disassembly of the 8-10S multimeric complex.
Oñate
S A
SA
Department of Pathology, University of Colorado Health Sciences Center, Denver 80262.
Estes
P A
PA
Welch
W J
WJ
Nordeen
S K
SK
Edwards
D P
DP
D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006360:Heat-Shock Proteins / Q000378:metabolism* / Q000502:physiology*; D006801:Humans; D011485:Protein Binding / Q000502:physiology; D011980:Receptors, Progesterone / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure
D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D011980:Receptors, Progesterone; D004247:DNA