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M1 muscarinic acetylcholine receptors activate zif268 gene expression via small G-protein Rho-dependent and
1Department of Neurochemistry, Faculty of Medicine, The University of Tokyo, Japan. thirabay@m.u-tokyo.ac.jp
Abstract:
We have previously shown that stimulation of M1 muscarinic acetylcholine receptors (mAChRs) in neuronal PC12D cells rapidly induces the immediate-early gene zif 268 [Ebihara, T. & Saffen, D. (1997) J. Neurochem. 68, 1001-1010]. Here we show that stimulation of M1 mAChRs in these cells activates four distal serum response elements (SREs) in the zif 268 promoter, and that this activation is strongly inhibited by Clostridium botulinum C3 exoenzyme (C3), which specifically inactivates the small G-protein Rho. Even with high doses of C3, however, a portion of the activation remains intact, indicating that stimulation of M1 mAChRs activates zif 268 SREs via Rho-dependent and Rho-independent pathways. Moreover, the Rho-independent activation of zif 268 SREs is inhibited by the dominant-negative form of the small G-protein Ras, suggesting that Rho-independent activation of zif 268 SREs is mediated by Ras. To determine if muscarinic agonists activate RhoA, we also measured the translocation of RhoA from the cytosolic fraction to the particulate fraction. Translocation of RhoA to the particulate fraction was observed within 15 min following stimulation of M1 mAChRs, indicating that RhoA is activated with sufficient rapidity to participate in the induction of zif 268 mRNA. Together, these results suggest that RhoA is activated following stimulation of M1 mAChRs and functions in SRE-dependent induction of the zif 268 gene within a Ras-independent pathway.
Insights
Stimulating M1 muscarinic acetylcholine receptors (mAChRs) activates the zif 268 gene through both Rho-dependent and Rho-independent pathways involving Ras. RhoA activation occurs rapidly, participating in zif 268 gene induction.
Area of Science:
- Neuroscience
- Molecular Biology
- Cell Signaling
Background:
- M1 muscarinic acetylcholine receptors (mAChRs) are known to induce the immediate-early gene zif 268 in PC12D cells.
- The precise signaling pathways downstream of M1 mAChR stimulation remain incompletely understood.
Purpose of the Study:
- To elucidate the signaling mechanisms by which M1 mAChR stimulation activates the zif 268 gene promoter.
- To investigate the roles of small G-proteins Rho and Ras in this activation process.
Main Methods:
- Stimulation of PC12D cells with M1 mAChR agonists.
- Analysis of zif 268 promoter activity using serum response elements (SREs).
- Inhibition studies using Clostridium botulinum C3 exoenzyme (C3) and dominant-negative Ras.
- Measurement of RhoA translocation via Western blotting.
Main Results:
- M1 mAChR stimulation activates four distal SREs in the zif 268 promoter.
- Activation is partially inhibited by C3, indicating Rho-dependent and Rho-independent pathways.
- Rho-independent activation is mediated by Ras.
- RhoA translocates to the particulate fraction within 15 minutes of M1 mAChR stimulation.
Conclusions:
- M1 mAChR stimulation activates zif 268 gene induction through both Rho-dependent and Rho-independent pathways.
- RhoA is rapidly activated and participates in the zif 268 gene induction.
- Ras mediates the Rho-independent component of zif 268 SRE activation.