The hormone gastrin helps maintain gastric mucosal integrity by regulating expression of genes such as plasminogen activator inhibitor 2 (PAI-2; 1) and regenerating protein 1 (Reg1; 2), but the mechanisms involved are incompletely understood. We recently identified a gastrin response element in the vesicular monoamine transporter 2 promoter and showed its activity depended on the proteasome beta subunit PSMB1 (3). We have used the gastric cancer cell line AGS-GR, which expresses the cholecystokinin 2 receptor (CCK2R), to determine if PSMB1 is involved in CCK2R-mediated expression of other gastrin sensitive genes, and to visualize its cellular distribution during gastrin stimulation. AGS-GR cells were transfected with siRNA against PSMB1 (30nM), or scrambled control, and 48h later with firefly luciferase reporter constructs containing 1.6kb of the PAI-2 promoter or 2.1kb of the Reg 1 promoter, together with a control renilla reporter vector. 18h later cells were stimulated with gastrin (G17, 2nM) or vehicle for 6h then extracted for dual luciferase assay. Western blots showed that 72h after transfection with PSMB1 siRNA, PSMB1 protein was reduced by 69.5±5.5% (mean±SEM, n=3) compared to the level in cells transfected with scrambled siRNA. In cells transfected with scrambled siRNA, gastrin increased transcription of the PAI-2 promoter 9.2±1.5 fold (n=6) and the Reg1 promoter 2.4±0.9 fold (n=9), compared to unstimulated cells. In cells with PSMB1 knocked down, gastrin-stimulated PAI-2 promoter activity was significantly reduced to 2.1±0.2 fold (p<0.001, ANOVA) and Reg1 activity to 1.2±0.1 fold (p<0.03). In unstimulated AGS-GR cells, immunocytochemistry using a rabbit antiserum against PSMB1 revealed distribution throughout the cytoplasm and nucleus. After 2h stimulation with 2nM G17, cytoplasmic PSMB1 was barely detectable and nuclear staining was intensified; after 6h stimulation, PSMB1 was again visible in cytoplasm. Western blotting of nuclear and cytoplasmic extracts showed that the nuclear:cytoplasmic ratio of PSMB1 changed from 1.7±0.4 in untreated cells to 10.6±2.6 (n=7, p<0.01, ANOVA) after 2h stimulation with G17. This subcellular redistribution was not seen with alpha (PSMA5) or regulatory (PSMC1) proteasome subunits. PSMB1 distribution was unaffected by activation of receptors for histamine, epidermal growth factor, PGE2, or interleukin-8. The response to G17 was however mimicked by phorbol 12-myristate 13-acetate (10-7M), and prevented by the protein kinase C (PKC) antagonist Ro-32-0432 (10-6M). We conclude that proteasome beta subunits are involved in gastrin-stimulated expression of some genes important for gastric mucosal integrity and function. CCK2R activation induces a PKC-dependent subcellular redistribution of proteasome subunits that may be linked to their transcriptional functions.