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

Abstract

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 M₃ 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 M₃ receptors to avoid differences in receptor phosphorylation influencing arrestin recruitment. We measured substantial differences in the agonist efficacies to induce M₃R-arrestin3 versus M₃R-GRK2 interaction. However, the rank order of the agonists for G protein- and GRK2-M₃R interaction was the same, suggesting that G protein and GRK2 binding to M₃R requires similar receptor conformations, whereas requirements for arrestin3 binding to M₃R are distinct.