Muscarinic acetylcholine receptors (mAChRs) belong to the family of G protein-coupled receptors (GPCRs). GPCR-induced signaling can be the result of the interplay between complex molecular events as several GPCRs can activate multiple different adaptor proteins. The M1-mAChR subtype couples preferentially to Gq proteins, but can also promiscuously stimulate Gs and Gi proteins [1]. cAMP is a second messenger which is mainly generated by adenylyl cyclases (ACs). The latter can be activated by Gs protein-independent signaling by an intracellular increase in Ca2+-ions. Gs proteins can either be activated as a direct response to receptor activation or through a crosstalk with activated Gq proteins [2]. In the present work, we investigated whether agonist-induced cAMP increase in CHO-M1 cells is due to Gs protein activation or to a Ca2+ increase in response to Gq protein activation. To this end, we employed acetylcholine, iperoxo, and carbachol and investigated agonist-induced signaling in CHO-M1 cells. To discern between Gq, Gs and Gi-dependent signaling, we applied Pertussis toxin (PTX) to block Gi proteins, Cholera toxin (CTX) to mask Gs-dependent signaling and FR900359 (FR) to block Gq proteins [3] or a combination of these toxins to detect the contribution of different classes of G proteins. The Gq inhibitor FR was able to decrease cAMP production in CHO-M1 cells, indicating that Gq proteins play an important role in AC activation. Strikingly, the FR effect on ACs could be attenuated by additional blocking of Gi proteins, although Gi proteins did not show any effect on cAMP modulation in CHO-M1 cells in case the Gq proteins are not silenced. To test whether the activation of ACs is due to an increase in intracellular Ca2+-ions or rather due to crosstalk between Gq and Gs proteins, we performed further cAMP assays by adding the cell-permeable Ca2+ chelator BAPTA-AM. We did not detect any effect of BAPTA-AM on AC activation, indicating no direct activation of ACs by Ca2+ under these conditions. We conclude that a direct crosstalk between Gq and Gs proteins may be the underlying mechanism for Gq-dependent AC activation in CHO-cells. 1. Thomas, R. et al.: J. Pharmacol. Exp. Ther. 2008, 327(2):365-74 2. Olianas, MC. et al.: Neurosignals 2013, 21:61-74 3. Schrage, R. et al.: Nat. Commun. 2015, 14;6:10156 doi: 10.1038/ncomms10156