Regulation of opioid tolerance by agonist-selective phosphorylation of endogenous µ-opioid-receptors in vivo

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Morphine is still one of the most effective drugs to treat severe pain. However, the clinical benefit of morphine and other opioid analgesics in the treatment of chronic pain is limited by the rapid development of tolerance and dependence. Morphine mediates all of its pharmacological effects via the G-protein-coupled mu-opioid receptor (MOR). Phosphorylation is an important mechanism for the regulation G protein-coupled receptors (GPCRs) mainly by altering their ability to interact with different intracellular partners. The agonist-induced phosphorylation of GPCRs is mediated by a family of G protein-coupled receptor kinases (GRKs) that specifically recognize the activated state of these receptors. We have previously shown that agonist-induced phosphorylation of MOR occurs at a conserved 10-residue sequence, 370TREHPSTANT379, in the carboxyl-terminal cytoplasmic tail. Morphine induces a selective phosphorylation of serine375 (S375) in the middle of this sequence that is predominantly catalyzed by GRK5. By contrast, high-efficacy opioids such as DAMGO or fentanyl not only induce phosphorylation of S375 but also drive higher-order phosphorylation on the flanking residues threonine370 (T370), threonine376 (T376), and threonine379 (T379) in a hierarchical phosphorylation cascade that specifically requires GRK2/3 isoforms. We have also shown that GRK2/3-mediated phosphorylation is responsible for rapid desensitization of MOR, while GRK5 is an essential element in the development of opioid dependence. The objectives of this proposal are 1) to analyze the agonist-selective phosphorylation of MOR in vivo using a novel HAMOR knockin mouse, 2) to elucidate the function of C-terminal phosphorylation in the development of opioid tolerance using phosphorylation-deficient MOR knockin mice, 3) to characterize the role of GRK5 in the development of opioid dependence using a novel GRK5-KD (kinase-dead) knockin mouse, and 4) to determine the contribution of heterologous PKC-mediated MOR phosphorylation to the development of morphine tolerance.