Stimulation of Cultured Cerebellar Granule Cells via Glutamate Receptors Induces TRE‐ and CRE‐Binding Activities Mediated by Common DNA‐Binding Complexes

Abstract: 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.