A Single-Stranded DNA Binding Site in the Human A1 Adenosine Receptor Gene Promoter

  1. Hongzu Ren and
  2. Gary L. Stiles
  1. Departments of Medicine and Pharmacology, Duke University Medical Center, Durham, North Carolina 27710

    Abstract

    Human A1 adenosine receptor gene expression is controlled by two independent promoters. The upstream promoter, promoter A, is subject to tissue specific regulation because not all cells express the mRNA associated with this promoter. One potential regulatory sequence located downstream of the TATA box is an AGG element appearing in a tandem repeat. In a previous study, transient transfection assays showed that mutations made in those AGG elements substantially reduced promoter activity. In the current study, DNase I footprinting indicated nuclear protein binding to this sequence between the TATA box and transcriptional start site. Electrophoretic mobility shift assay confirmed further the presence of an AGG element binding protein (AGBP) in human brain nuclear protein extracts. This binding protein has much higher affinity for single-stranded than for double-stranded DNA, and the binding is sequence specific. A series of assays also showed that AGBP is not related to the nuclear factor SP1 and the binding does not require metal cofactors. Therefore, AGBP is likely to be a specific single-stranded DNA binding protein that is required for the full expression of A1 adenosine receptor gene and particularly abundant in brain tissue.

    Footnotes

    • Send reprint requests to: Gary L. Stiles, M.D., Duke University Medical Center, Box 3444, Durham, NC 27710 E-mail:stiles{at}hodgkin.mc.duke.edu

    • G.L.S. was supported by a National Heart, Lung and Blood Institute SCOR Grant (5P50HL54314) in Ischemic Disease.

    • Abbreviations:
      AGBP
      AGG element binding protein
      EMSA
      electrophoretic mobility shift assay
      -F
      coding sequence
      -R
      noncoding sequence
      EGTA
      ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
      HEPES
      4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
      EGF-r
      epidermal growth factor receptor
      DTT
      dithiothreitol
      WT
      wild-type
      • Received March 27, 1997.
      • Accepted September 22, 1997.
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    1. Molecular Pharmacology January 1, 1998 vol. 53 no. 1 43-51
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