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GTP-binding proteins and adenylate cyclase activity in v-Ki-ras transformed NIH/3T3 fibroblast cells
L Konishi-Imamura1, M Tohda, M Noda
1Department of Pharmacology, Toyama Medical and Pharmaceutical University, Japan.
Abstract:
To identify the role of ras oncogene and p21 in the coupling mechanism of GTP-binding proteins to adenylate cyclase, we used v-Ki-ras transformed NIH/3T3 fibroblast cells. In the previous study, we investigated that NaF, cholera toxin and forskolin remarkably enhanced the adenylate cyclase activity in transformed cells compared to normal NIH/3T3 cells. In the present study, adenylate cyclase was more enhanced by GTP gamma S in transformed cells than in normal cells. It was considered that p21 plays enhancing role in coupling of GTP-binding proteins to adenylate cyclase. Further, as measured by the degree of [32P] ADP-ribosylation of GTP-binding proteins by cholera toxin and pertussis toxin respectively, the amount of Gs (46 kDa) was almost equal in both cells, while the amount of Gi (41 kDa) in transformant was about one third of that in normal cells. This difference seems to be reflected in either the biological situations or the quantities of Gi. Our data suggest that v-Ki-ras transformation resulted in the decrease of Gi protein so that the inhibitory regulation on adenylate cyclase relatively becomes low and then stimulatory influence of Gs seems to be enhanced.
Insights
Ras oncogene p21 enhances GTP-binding protein coupling to adenylate cyclase. v-Ki-ras transformation decreases inhibitory Gi protein, enhancing Gs-mediated stimulation.
Area of Science:
- Molecular Biology
- Cellular Signaling
- Oncology
Background:
- Ras oncogene and p21 are implicated in cellular signaling pathways.
- Previous studies showed enhanced adenylate cyclase activity in ras-transformed cells.
- GTP-binding proteins (G proteins) regulate adenylate cyclase activity.
Purpose of the Study:
- To elucidate the role of ras oncogene and p21 in G protein-adenylate cyclase coupling.
- To investigate the impact of v-Ki-ras transformation on G protein levels and activity.
Main Methods:
- Utilized v-Ki-ras transformed NIH/3T3 fibroblast cells and normal NIH/3T3 cells.
- Assessed adenylate cyclase activity using GTP gamma S.
- Quantified G protein levels (Gs and Gi) via [32P] ADP-ribosylation assays with cholera toxin and pertussis toxin.
Main Results:
- GTP gamma S more significantly enhanced adenylate cyclase in transformed cells.
- Gs protein levels were comparable between normal and transformed cells.
- Gi protein levels were reduced by approximately one-third in v-Ki-ras transformed cells compared to normal cells.
Conclusions:
- p21 associated with ras oncogene likely enhances the coupling of GTP-binding proteins to adenylate cyclase.
- v-Ki-ras transformation leads to decreased Gi protein levels.
- Reduced Gi protein results in diminished inhibitory regulation of adenylate cyclase, enhancing the stimulatory influence of Gs.