Disentangling bias between Gq, GRK2, and arrestin3 recruitment to the M3 muscarinic acetylcholine receptor

G protein-coupled receptors (GPCRs) transmit extracellular signals to the inside by activation of intracellular effector proteins. Different agonists can promote differential receptor-induced signaling responses – termed bias – potentially by eliciting different levels of recruitment of effector proteins. As activation and recruitment of effector proteins might influence each other, thorough analysis of bias is difficult. Here, we compared the efficacy of seven agonists to induce G protein, G protein-coupled receptor kinase 2 (GRK2), as well as arrestin3 binding to the muscarinic acetylcholine receptor M 3 by utilizing FRET-based assays. In order to avoid interference between these interactions, we studied GRK2 binding in the presence of inhibitors of G i and G q proteins and analyzed arrestin3 binding to prestimulated M 3 receptors to avoid differences in receptor phosphorylation influencing arrestin recruitment. We measured substantial differences in the agonist efficacies to induce M 3 R-arrestin3 versus M 3 R-GRK2 interaction. However, the rank order of the agonists for G protein- and GRK2-M 3 R interaction was the same, suggesting that G protein and GRK2 binding to M 3 R requires similar receptor conformations, whereas requirements for arrestin3 binding to M 3 R are distinct.