Stimulation of cultured cerebellar granule cells via glutamate receptors induces TRE- and CRE-binding activities mediated by common DNA-binding complexes.
By use of nuclear mini-extracts prepared from cultured cerebellar granule cells in a gel-mobility assay, exogenous N-methyl-D-aspartate (NMDA) or kainate was shown to increase both 12-O-tetradecanoylphorbol 13-acetate-responsive element (TRE)- and cyclic AMP-responsive element (CRE)-binding activity. These increases were specifically prevented by the NMDA receptor antagonist D,L-2-amino-5-phosphonovalerate and the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, respectively. The increase of TRE-binding activity was dependent on de novo protein synthesis, and its inductions by both NMDA and kainate required extracellular Ca2+. TRE-binding activity was competitively inhibited by the CRE, and vice versa, showing higher DNA-binding affinity to the CRE than to the TRE. A proteolytic clipping bandshift assay demonstrated that the increase in CRE-binding activity could be mediated by the TRE-binding activity. Thus, the TRE-binding activity cross-binding to the CRE could be activated by NMDA or kainate stimulation. The involvement of c-Fos or Fos-related proteins in the TRE- and CRE-binding complexes was shown by a supershift gel-mobility assay using anti-c-Fos antiserum.
Department of Microbiology, Faculty of Pharmaceutical Sciences, Okayama University, Japan.
H
H
Sakurai
R
R
Kurusu
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K
Sano
T
T
Tsuchiya
M
M
Tsuda
D015763:2-Amino-5-phosphonovalerate / Q000494:pharmacology; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002118:Calcium / Q000494:pharmacology; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology* / Q000187:drug effects / Q000648:ultrastructure; D000242:Cyclic AMP / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000502:physiology*; D007608:Kainic Acid / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D016202:N-Methylaspartate / Q000494:pharmacology; D011485:Protein Binding; D016760:Proto-Oncogene Proteins c-fos / Q000502:physiology; D011810:Quinoxalines / Q000494:pharmacology; D017470:Receptors, Glutamate / Q000187:drug effects / Q000502:physiology*; D016194:Receptors, N-Methyl-D-Aspartate / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism*
D004268:DNA-Binding Proteins; D016760:Proto-Oncogene Proteins c-fos; D011810:Quinoxalines; D017470:Receptors, Glutamate; D016194:Receptors, N-Methyl-D-Aspartate; D016202:N-Methylaspartate; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione; D015763:2-Amino-5-phosphonovalerate; D000242:Cyclic AMP; D008274:Magnesium; D013755:Tetradecanoylphorbol Acetate; D007608:Kainic Acid; D002118:Calcium