This proposal focuses on the molecular mechanisms of opioid receptors in intact neural cells and in the lipid environment of the plasma membrane. One goal is to identify regulatory processes of opioid signal transduction in a given cell in which multiple Opioid and non-opioid receptors function concurrently, sharing thereby transducer and effector proteins (""""""""cross- talk""""""""). This work will characterize those processes or components of receptor signaling that will become rate-limiting in the concerted environment of intact cells, and reveal mechanisms of receptor adaptation. Applying multiple methodological approaches (including quantitation of ligand-receptor-transducer-effector interactions, covalent protein modification, use of antibodies and antisense oligonucleotides, immunoblotting, receptor over-expression, cell membrane/liposome fusion/reconstitution with purified protein components, enzyme assays, ion transport), the selectivity, stoichiometry and regulation of coupling of mu and delta opioid receptors to type-specific adenylyl cyclase and Ca2+- channels through G protein subtypes will be assessed, including the role of cAMP-dependent protein kinase. Subsequently, the mutual modulation between the two opioid and an opioid and inhibitory adrenergic and muscarinic receptor systems will be studied in cells in which the concentration/availability of receptor/transducer/effector is selectively altered to induce adaptation. To understand the significance of trophic factors and of cell-to-cell communication occurring in brain, opioid signal transduction will also be studied under conditions of neuron-glia interaction in vitro, and in cells in which receptor function is influenced by differentiating agents. In neural cells exhibiting biochemical correlates (cAMP levels) of opioid tolerance and dependence, the regulation of receptor and of selective G protein content and coupling, and the role Of G protein selectivity will be evaluated. In conjunction, the contribution of receptor reserve and of receptor-G protein coupling to agonist potency and efficacy in eliciting an effector response (adenylyl cyclase, Ca2+ transport) will be assessed. Within the overall focus on regulation by molecular cross-talk, the other goal is to characterize the mechanisms underlying the potent modulation o opioid receptor function (conformation, binding affinity/capacity, membrane dynamics) by membrane lipids and by the biophysical property of the membrane environment. Using lipid transfer proteins and fusion techniques for membrane modification, fluorescent opioids and G proteins, and fluorescent techniques of high sensitivity and resolution (time-resolved fluorescence and fluorescence recovery after photobleaching), the functionally essential lipid boundary layer around opioid receptors will be identified, the mobility of receptor and G protein in the plasma membrane determined and its role in the regulation of opioid signal transduction assessed (collision-coupling mechanism). In summary, the proposed research describes the use of advanced approaches of biochemistry, pharmacology, and neuroscience to elucidate the regulation of opioid function at the molecular, membrane, and cellular level.
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