The μ-opioid receptor (MOR) is a G-protein-coupled receptor (GPCR) that mediates both analgesic effects and adverse effects of opioid drugs. Despite extensive efforts to develop a signal-biased drug, drugs with sufficiently reduced side effects have not been established, in part owing to lack of comprehensive signal transducer profiles of MOR. In this study, by profiling the activity of signal transducers including G proteins and GPCR kinases (GRKs), we revealed an unprecedented mechanism of selective GRK3 activation by Gβ5, leading to β-arrestin recruitment. By utilizing multiple genome-edited cell lines and functional assays, we found that oliceridine, an FDA-approved G-protein-biased agonist, selectively activates Gαz- and GRK3-mediated signaling. Notably, among the five Gβ subtypes, only Gβ5 distinguishes GRK3 from GRK2. Using single-molecule imaging, we found that GRK3 is recruited to the plasma membrane upon MOR agonist stimulation by Gβ1 and Gβ5, yet their interaction dynamics with GRK3 and mechanisms of action are different. Furthermore, particle diffusion analysis suggests that Gβ5 is enriched in confined membrane domains, through which GRK3 is recruited to the plasma membrane in a freely diffusible state, thereby allowing GRK3 to efficiently interact with MOR. These findings provide a mechanism by which MOR agonists rely on a specific Gα-Gβ-GRK axis to induce β-arrestin recruitment.
Keywords: G proteins; G-protein-coupled receptor; GPCR kinase (GRK); Single-molecule analysis; β-arrestin; μ-opioid receptor.
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