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J Biol Chem, Vol. 273, Issue 42, 27474-27483, October 16, 1998

Sp1 and Sp3 Regulate Transcriptional Activity of the Facilitative Glucose Transporter Isoform-3 Gene in Mammalian Neuroblasts and Trophoblasts

Rosario A. Rajakumar, Shanthie Thamotharan, Ram K. Menon^§, and Sherin U. Devaskar

From the  Divisions of Neonatology and Developmental Biology and ^§ Endocrinology, the Department of Pediatrics, University of Pittsburgh School of Medicine, Magee-Womens Research Institute, Pittsburgh, Pennsylvania 15213

The murine facilitative glucose transporter isoform 3 (Glut 3) is developmentally regulated and is predominantly expressed in neurons and trophoblasts. Employing the primer extension and RNase protection assays, the transcription start site (denoted as +1) of the murine Glut 3 gene was localized to 305 base pairs (bp) 5' to the ATG translation start codon. Transient transfection assays in N2A, H19-7 neuroblasts, and HRP.1 trophoblasts using sequential 5'-deletions of the murine Glut 3-luciferase fusion gene indicated that the 203 to +237 bp region with reference to the transcriptional start site contained promoter activity. Repressor function was limited to the 137 to 130 bp region within the transcriptional activation domain. The nuclear factors Sp1 and Sp3 bound this GC-rich region in N2A, H19-7, and HRP.1 cells. Dephosphorylation of Sp1 was essential for Glut 3 DNA binding. The related Sp3 protein also bound this same region of mouse Glut 3 in all three cell lines. Mutations of the Sp1-binding site employed in transient transfection and mobility shift assays confirmed the nature of the DNA-binding proteins, while supershift assays with anti-Sp1 and anti-Sp3 IgGs characterized the differences in the two DNA-binding proteins. Co-transfection of the Glut 3-luciferase fusion gene with or without mutations of the Sp1-binding site along with the Sp1 or Sp3 expression vectors in Drosophila SL2 cells confirmed a reciprocal effect, with Sp1 suppressing and Sp3 activating Glut 3 gene transcription.

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