A 26-amino acid insertion domain defines a functional transcription switch motif in Pit-1beta

Pit-1, a pituitary-specific POU homeodomain transcription factor, specifies three anterior pituitary lineages; governs growth hormone, prolactin, and thyrotropin gene expression; and mediates basal and Ras-stimulated prolactin promoter activity in GH4 pituitary cells. Alternate splicing of the Pit-1 message produces the Pit-1beta isoform, which contains a 26-amino acid insertion, the beta-domain, within the amino-terminal transactivation domain. The beta-domain functions as a molecular switch, such that Pit-1beta blocks both basal and Ras-stimulated prolactin promoter activity in GH4 pituitary cells yet preferentially enhances protein kinase A-stimulated prolactin promoter activity in a HeLa reconstitution system. To determine whether the amino acid sequence of the beta-domain dictates function, we replaced it with five different 26-amino acid sequences. These mutants fail to block basal or Ras-stimulated rat prolactin promoter activity and fail to optimally enhance the protein kinase A response of prolactin promoter. These data demonstrate that the amino acid sequence of the beta-domain specifies its role as a molecular switch. Additionally, the presence of both Pit-1 and Pit-1beta in pituitary cells allows diverse incoming signals to utilize structurally different forms of the same gene product, which can interact with distinct co-factors, integrating multiple signaling pathways at the level of the nucleus.