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An oestrogen receptor activator protein in rat uterine cytosol
Abstract:
Earlier reports of oestrogen receptor binding to DNA indicated that a protein which does not bind oestrogen might be involved in the conversion of the 4S oestradiol receptor to the 5S form but no direct evidence for the existence of this protein was presented. This receptor transformation or activation step is thought to be a requirement for events in the nucleus. We considered it likely that such a protein might be similar to the cytoplasmic proteins which stimulate eukaryotic RNA polymerases. These are basic proteins which are not adsorbed to DEAE cellulose and have a sedimentation coefficient of approximately 3S. To examine this possibility, we fractionated unlabelled uterine cytosol by DEAE-cellulose chromatography. Unlabelled cytosol was used to avoid the possibility that such a factor might be retained on the column as a component of the receptor--oestradiol complex. Here we present evidence of a protein present in uterine cytosol which forms a complex with the 4S form of the oestrogen receptor to produce a 5S-activated form. This activated form binds to uterine nuclei, DNA-cellulose and native calf thymus DNA. The cytosol protein, which we call receptor activation factor, does not bind oestradiol, has a sedimentation coefficient of approximately 3S and does not bind to DEAE-cellulose.
Insights
Researchers discovered a new protein, receptor activation factor, in uterine cytosol. This protein converts the 4S oestrogen receptor to a 5S form, enabling it to bind to uterine nuclei and DNA.
Area of Science:
- Molecular Biology
- Cell Biology
- Endocrinology
Background:
- Oestrogen receptor (ER) binding to DNA is crucial for gene regulation.
- A transformation from a 4S to a 5S receptor form is believed necessary for nuclear activity.
- Previous studies suggested a non-oestrogen-binding protein might mediate this transformation, but lacked direct evidence.
Purpose of the Study:
- To identify and characterize the putative protein involved in oestrogen receptor activation.
- To investigate the mechanism by which the 4S oestrogen receptor is converted to a DNA-binding 5S form.
Main Methods:
- Fractionation of unlabelled uterine cytosol using DEAE-cellulose chromatography.
- Characterization of protein properties including sedimentation coefficient and binding affinities.
- Assessment of the interaction between the isolated protein and the oestrogen receptor.
Main Results:
- Evidence presented for a novel protein in uterine cytosol, termed receptor activation factor (RAF).
- RAF forms a complex with the 4S oestrogen receptor, generating a 5S activated form.
- The 5S activated receptor complex exhibits binding to uterine nuclei, DNA-cellulose, and native calf thymus DNA.
- RAF itself does not bind oestradiol, possesses a sedimentation coefficient of approximately 3S, and does not bind to DEAE-cellulose.
Conclusions:
- Receptor activation factor is a distinct protein that facilitates the transformation of the oestrogen receptor to a DNA-binding state.
- This activation step is essential for the oestrogen receptor's nuclear function.
- The findings provide direct evidence for a protein mediator of oestrogen receptor activation and nuclear translocation.