Identification of a regulatory sequence within the third intracellular loop that governs β-arrestin binding to the muscarinic M5 receptor

The M5 muscarinic acetylcholine receptor (M5R) is selectively expressed in dopaminergic neurons and although accounts for <2 % of the total muscarinic receptor population, plays critical roles in many physiological and pathophysiological processes. Despite its undoubted importance, the M5R remains greatly understudied. This study aimed to elucidate the regulatory mechanisms underlying M5R signaling, particularly in relation to G-protein-coupled receptor kinases (GRKs), β-arrestin recruitment, and internalization. Utilizing a combination of BRET based assays, flow cytometry and biochemical techniques we demonstrate that, following ligand binding and G-protein signaling, M5R undergoes GRK2/3-mediated, β-arrestin1/2 recruitment followed by β-arrestin1/2 and clathrin-dependent internalization. We used a series of deletion mutants to identify two key regions within the third intracellular loop critical for increasing and decreasing β-arrestin recruitment, respectively. Notably, we identified amino acids 303–345 as crucial for recruitment of β-arrestins, the deletion of which results in a G-protein-biased M5R ∆1–2. Using this receptor mutant in conjunction with β-arrestin1/2 CRISPR/Cas9 edited cells, we show M5R activation of extracellular signal-regulated kinase (ERK1/2) phosphorylation and whole-cell responses require G-protein-dependent initiation, however, phosphorylation and β-arrestin contribute to the spatial regulation of signaling.