An alternatively spliced Pit-1 isoform altered in its ability to trans-activate.
Although alternative splicing has been shown to give rise to isoforms of a number of transcription factors, such isoforms have not previously been detected for the POU homeodomain protein Pit-1. Screening of a rat pituitary GH3 cell cDNA expression library yielded a clone, termed pCMVPit-1a, encoding a 35.8 kD protein (Pit-1a) containing a 26 amino acid insert in the Pit-1 trans-activation domain. The position of the insert, plus Southern blot analysis, implied that Pit-1a mRNA arises by alternative splicing of the Pit-1 gene transcript. Pit-1a mRNA was detected in GH3 rat pituitary tumor cells at levels about 1/7 that of Pit-1 mRNA. Pit-1a mRNA-specific sequences were also detected in rat and mouse pituitary, and in mouse thyrotropic tumor TtT cells. DNA mobility shift assays showed that Pit-1a binds specifically to Pit-1 binding sites in the proximal prolactin promoter, but produces DNA-protein complexes of markedly different mobilities than Pit-1. In stably transfected CHO cells which accumulated approximately equal levels of either of the two proteins, Pit-1 trans-activated a prolactin promoter-driven CAT construct, while Pit-1a yielded no detectable transactivation, implying a trans-activation ratio for Pit-1a/Pit-1 of less than 0.05. Thus, the insertion of 26 amino acids of similar composition into the activation domain of Pit-1 has at once affected both the mode of binding of this protein and its ability to function as a trans-activator.
Morris
A E
AE
Department of Biological Sciences, Columbia University, New York, NY 10027.
Kloss
B
B
McChesney
R E
RE
Bancroft
C
C
Chasin
L A
LA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015139:Blotting, Southern; D015153:Blotting, Western; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009928:Organ Specificity / Q000235:genetics; D010902:Pituitary Gland / Q000378:metabolism; D011388:Prolactin / Q000235:genetics; D011401:Promoter Regions, Genetic; D012326:RNA Splicing; D051381:Rats; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection
D004268:DNA-Binding Proteins; C494187:POU1F1 protein, human; C494186:Pou1f1 protein, rat; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; D011388:Prolactin; D004247:DNA