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J Biol Chem ; 277(29): 25877-83, 2002 Jul 19.
Article in English | MEDLINE | ID: mdl-11943774

ABSTRACT

The beta-subunit of cGMP-phosphodiesterase (beta-PDE) is a key protein in phototransduction expressed exclusively in rod photoreceptors. It is necessary for visual function and for structural integrity of the retina. beta-PDE promoter deletions showed that the -45/-23 region containing a consensus Crx-response element (CRE) was necessary for low level transcriptional activity. Overexpressed Crx modestly transactivated this promoter in 293 human embryonic kidney cells; however, mutation of CRE had no significant effect on transcription either in transfected Y79 retinoblastoma cells or Xenopus embryonic heads. Thus, Crx is unlikely to be a critical beta-PDE transcriptional regulator in vivo. Interestingly, although the beta/GC element (-59/-49) binds multiple Sp transcription factors in vitro, only Sp4, but not Sp1 or Sp3, significantly enhanced beta-PDE promoter activity. Thus, the Sp4-mediated differential activation of the beta-PDE transcription defines the first specific Sp4 target gene reported to date and implies the importance of Sp4 for retinal function. Further extensive mutagenesis of the beta-PDE upstream sequences showed no additional regulatory elements. Although this promoter lacks a canonical TATA box or Inr element, it has the (T/A)-rich beta/TA sequence located within the -45/-23 region. We found that it binds purified TBP and TFIIB in gel mobility shift assays with cooperative enhancement of binding affinity.


Subject(s)
3',5'-Cyclic-GMP Phosphodiesterases/genetics , Homeodomain Proteins/metabolism , Phosphoric Diester Hydrolases , Promoter Regions, Genetic , Retinal Rod Photoreceptor Cells/enzymology , Trans-Activators/metabolism , Transcription Factors/metabolism , Animals , Base Sequence , Cyclic Nucleotide Phosphodiesterases, Type 6 , DNA-Binding Proteins/metabolism , Humans , Molecular Sequence Data , Sp4 Transcription Factor , TATA-Box Binding Protein , Transcription Factor TFIIB , Transfection , Tumor Cells, Cultured , Xenopus
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