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.
Department of Medicine, Program in Molecular Biology and Colorado Cancer Center, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA. a.gutierrez-hartmann@UCHSC.edu
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D000595:Amino Acid Sequence; D000818:Animals; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D050783:Proto-Oncogene Proteins c-ets; D051381:Rats; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; C494187:POU1F1 protein, human; C494186:Pou1f1 protein, rat; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D050817:Transcription Factor Pit-1; D014157:Transcription Factors