Identification of Protein Kinase C Targets Leading to Opioid Tolerance

Prolonged treatment with opiates, the most widely used analgesics, leads to the development of tolerance and addiction. High-efficacy opiate agonists such as fentanyl activate the morphine receptor (MOR) leading to receptor translocation throughout the plasma membrane and phosphorylation by G protein receptor kinases, followed by receptor clustering to microdomains and transient nuclear ERK activation preceding receptor endocytosis and resensitization. Low-efficacy agonists such as morphine activate MOR leading to receptor phosphorylation by localized plasma membrane associated protein kinase C (PKC) and a restricted redistribution of MOR with sustained cytosolic ERK activation. Genetic and pharmacological inhibition of PKC in rodents has demonstrated PKC to be a key player responsible for morphine tolerance and addiction. The molecular mechanisms that lead to PKC mediated tolerance still remain to be elucidated. Dissecting PKC signaling is difficult due to the overlapping specificity of different members of the PKC family of kinases and the fact that kinase substrate interactions are normally transient. To this end, in the present project we intend to use a combination of novel tools (activation specific antibodies, biotin ligase fused PKC, & PKC substrate trapping mutants) and modern proteomics approaches to identify PKC substrates and binding partners involved in morphine induced tolerance both in cell culture systems and in animal models. The specific aims are: to use active state specific anti cPKC antibodies to identify PKC binding proteins (by mass spectrometry) and determine the dynamics of PKC activation in mice following morphine treatment (Aim 1), to employ the BioID method to identify PKC targets (by mass spectrometry) upon morphine induced MOR activation (Aim 2), and to utilize PKC substrate trapping mutants to identify targets (by mass spectrometry) upon morphine induced MOR activation (Aim 3). Together, the identification of key components involved in PKC mediated tolerance to morphine will contribute to the elucidation of mechanisms underlying morphine tolerance and the development of novel therapeutics for the treatment of tolerance and addiction.