A novel G protein-biased agonist at the μ opioid receptor induces substantial receptor desensitisation through G protein-coupled receptor kinase

Background and Purpose G protein-biased mu opioid receptor agonists have the potential to induce less receptor desensitisation and tolerance than balanced opioids. Here, we investigated if the cyclic endomorphin analogue Tyr-c[D-Lys-Phe-Tyr-Gly] (Compound 1) is a G protein-biased mu agonist and characterised its ability to induce rapid receptor desensitisation in mammalian neurones. Experimental Approach The signalling and trafficking properties of opioids were characterised using bioluminescence resonance energy transfer assays, enzyme-linked immunosorbent assay and phosphosite-specific immunoblotting in human embryonic kidney 293 cells. Desensitisation of opioid-induced currents were studied in rat locus coeruleus neurones using whole-cell patch-clamp electrophysiology. The mechanism of Compound 1-induced mu receptor desensitisation was probed using kinase inhibitors. Key Results Compound 1 has similar intrinsic activity for G protein signalling as morphine. As predicted for a G protein-biased mu agonist, Compound 1 induced minimal agonist-induced internalisation and phosphorylation at intracellular mu receptor serine/threonine residues known to be involved in G protein-coupled receptor kinase (GRK)-mediated desensitisation. However, Compound 1 induced robust rapid mu receptor desensitisation in locus coeruleus neurons, to a greater degree than morphine. The extent of Compound 1-induced desensitisation was unaffected by activation or inhibition of protein kinase C (PKC) but was significantly reduced by inhibition of GRK. Conclusion and Implications Compound 1 is a novel G protein-biased mu agonist that induces substantial rapid receptor desensitisation in mammalian neurons. Surprisingly, Compound 1-induced desensitisation was demonstrated to be GRK dependent despite its G protein bias. Our findings refute the assumption that G protein-biased agonists will evade receptor desensitisation and tolerance.