The rabbit αs1-casein gene contains a distal prolactin-dependent enhancer 3442-3285 bp 5′ to the site of initiation of transcription. We have reported previously that four DNA/protein-binding sites (F1–F4) are located within this distal enhancer. We now show that one of this binding site (the F4 site) binds in vitro a MGF/STAT5-like factor. The functional importance of the F4 site was estimated by cotransfection of CHO cells with a chimeric gene containing or not the F4 sequence linked to the (−391/+1774)CAT gene and a plasmid encoding the rabbit mammary prolactin receptor. The F4 site is necessary for maximal response of the enhancer to prolactin. However, this site has to be associated to the F1–F3 fragment. It can be replaced by a genuine MGF/STAT5-binding site. A mutational analysis indicates that F4 and F1 sites are simultaneously involved to confer a high prolactin sensitivity.
