Downstream Signaling of Muscarinic M₄ Receptors Is Regulated by Receptor Density and Cellular Environment

Multiple muscarinic M₄ receptor modulators are currently advancing in clinical development for the treatment of positive symptoms in schizophrenia, including agonists and positive allosteric modulators. Considering the importance of comprehending M₄ receptor pharmacology for these therapeutic applications, this study investigates M₄ receptor signaling pathways upon activation by structurally diverse muscarinic agonists, exploring the role of receptor expression levels and cellular environment on downstream signaling. HEK-293 cells and rat primary neurons expressing human M₄ receptors were used to measure the kinetics of cAMP levels and compound effects on neuronal network activity. Receptor expression levels were controlled by a Tet-On system and quantified using a radioactive binding assay. Our findings revealed that most agonists caused a concentration-dependent reduction of cAMP levels (G_i/o) at low concentrations, while inducing an increase in cAMP at higher concentrations (G_s). A less prominent coupling via G_s was observed when receptor density in HEK-293 cells was reduced. In the neuronal assay, most compounds showed consistent inhibition of neuronal activity. A distinct group of agonists displayed a specific profile, with no G_s coupling at high receptor density, partial activation at low receptor density, and low to no effects in the neuronal assay. This study provides a side-by-side comparison of the activity of structurally diverse M₄ agonists and highlights compound-specific activation of GPCR intracellular signaling pathways. The data offer new insights into M₄ receptor pharmacology that may aid in the development of novel therapies for the treatment of psychiatric diseases.