M1 muscarinic acetylcholine receptors activate zif268 gene expression via small G-protein Rho-dependent and λ-independent pathways in PC12D cells

We have previously shown that stimulation of M-1 muscarinic acetylcholine receptors (mAChRs) in neuronal PC12D cells rapidly induces the immediate-early gene zif268 [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 zif268 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 zif268 SREs via Rho-dependent and Rho-independent pathways. Moreover, the Rho-independent activation of zif268 SREs is inhibited by the dominant-negative form of the small G-protein Ras, suggesting that Rho-independent activation of zif268 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 M-1 mAChRs, indicating that RhoA is activated with sufficient rapidity to participate in the induction of zif268 mRNA. Together, these results suggest that RhoA is activated following stimulation of M-1 mAChRs and functions in SRE-dependent induction of the zif268 gene within a Ras-independent pathway.