Mechanism of PKC-mediated desensitization of the morphine-activated mu opioid receptor in neurones

Repeated administration of opioid analgesic drugs such as morphine results in the development of tolerance whereby to obtain the same level of response increasing doses of the drug must be administered. Morphine produces it profound behavioural effects (analgesia, respiratory depression and euphoria) by activating specific proteins (receptors) located on the outer membrane of nerve cells in the brain and spinal cord. Prolonged activation of these receptors results in them being switched off (desensitized) and this desensitization may be a major component of tolerance. We have recently provided evidence that when activated by morphine, the mechanism of receptor desensitization involves a protein called protein kinase C, which switches off morphine signalling by a process called phosphorylation. However, we do not at present understand how phosphorylation by protein kinase C triggers this switching off in response to morphine. In this application, we propose to determine exactly how protein kinase C phosphorylation switches off morphine signalling in nerve cells. In particular, we will determine in the nerve cells whether or not it is protein kinase C-mediated phosphorylation of the receptor itself or another protein which mediates the desensitization. To do this we will express morphine receptors in nerve cells that have been altered to remove potential sites on the receptor that are phosphorylated by protein kinase C. Given that protein kinase C phosphorylation appears to be a crucial component of tolerance to morphine and related drugs, it is very important that we now determine the exact mechanism involved. In this project we aim to achieve this so that in the future new approaches can be developed to overcome the problems associated with morphine use and abuse.

Repeated administration of opiate drugs such as morphine results in the development of tolerance to their analgesic and euphoric effects. Tolerance can limit the clinical efficacy of opiate drugs and enhance the social problems inherent in recreational opioid abuse, but surprisingly the mechanism(s) underlying the development of tolerance remain controversial. Morphine exerts its biological effects by activating the mu-opioid receptor (MOR) and tolerance to the drug is likely to be due in part to desensitization of these receptors. We have obtained convincing evidence from both in vitro and in vivo studies that ongoing protein kinase C (PKC) activation is essential in order to observe morphine desensitization and tolerance. However, the molecular mechanism underlying PKC?s effect has not been determined. The central aim of this proposal is to identify in neurones the mechanism by which PKC activation mediates morphine-induced desensitization of MORs. PKC-mediated MOR desensitization in response to morphine could involve direct phosphorylation of MORs by PKC, or alternatively PKC phosphorylation of key regulatory proteins (e.g. G protein coupled receptor kinases) that mediate MOR desensitization. Here we will distinguish between these possibilities by applying viral technology to express, in neurones in primary culture, wild type MORs as well as MOR mutants lacking amino acid residues that are potential substrates for PKC phosphorylation. We will determine the ability of these MOR constructs to undergo PKC-mediated phosphorylation, as well as their ability to undergo morphine-induced desensitization by assessing their coupling to G protein-coupled Inwardly Rectifying K+ channels (GIRKs). We will then go on to identify the molecular mechanism underlying PKC?s involvement in morphine-induced MOR desensitization. Using a combination of co-immunoprecipitation and Fluorescence Resonance Energy Transfer (FRET) technology, we will determine in neurones whether PKC phosphorylation modifies the interaction between MORs and signalling proteins (i.e. G proteins) or regulatory proteins (e.g. G protein-coupled receptor kinases). Together these experiments will determine the molecular mechanism by which PKC mediates morphine-induced desensitization of MORs in neurones, and so identify what is likely to represent a crucial component of tolerance to morphine in the whole organism.