Following prolonged agonist activation, G protein-coupled receptors (GPCRs) become desensitised to prevent excessive stimulation. Desensitisation is usually triggered by receptor phosphorylation, either by G protein-coupled receptor kinases (GRKs) or by second messenger-dependent protein kinases (e.g. PKC). Following phosphorylation by GRKs, arrestins bind to the receptor, which uncouples the GPCR from its G protein¹. Arrestins also target the receptor for internalisation via clathrin-coated pits, following which the receptor is either dephosphorylated and recycled back to the cell surface, or it is targeted to lysosomes for degradation. We have used a combination of heterologous expression of receptor and analysis of endogenous receptors in mature neurones to identify the mechanisms underlying morphine-induced desensitisation of μ-opioid receptors (MOR). There is still much controversy about whether morphine actually induces significant MOR desensitisation in neurones, and by what mechanism, and also whether or not these molecular events bear any relationship to the important problem of morphine tolerance. For our heterologous expression system, we have stably transfected HEK293 cells with the G protein-coupled inwardly rectifying K⁺ (GIRK) channel subunits Kir3.1 and Kir3.2A and then transiently transfected them with MOR. The evoked current thus provides a real-time readout of receptor activation. Following addition of saturating concentrations of agonist, the evoked GIRK current reaches a peak and then declines (desensitises) in the continued presence of drug. The t_0.5 for both morphine and DAMGO (a peptide MOR agonist)-induced desensitisation was rapid at 1-2 min, and the extent of desensitisation was the same for the two agonists (~70%). However, the mechanism of desensitisation was different; DAMGO-induced desensitisation was blocked by a Dominant Negative Mutant construct of GRK2 (DNM GRK2), whilst morphine-induced desensitisation was unaffected by DNM GRK2, but was instead reduced by inhibitors of PKC². Thus in a heterologous system, agonists at the same GPCR that induce the same cellular response can undergo desensitisation by different mechanisms. Crucially, we have now shown that agonist-selective mechanisms of MOR desensitisation also occurs with endogenous MORs in mature neurones. Using rat brain slices containing locus coeruleus (LC) neurones, DAMGO induces rapid MOR desensitisation, whilst morphine can only do so (t_0.5 ~ 3 min) when PKC activity in the neurones is elevated, such as by co-activation of neuronal M2 muscarinic receptors with oxotremorine³. By injecting an adenoviral construct expressing DNM GRK2 into LC, we have shown that DAMGO-induced desensitisation in LC neurones is GRK-mediated, whilst the construct did not affect morphine plus PKC-mediated desensitisation. Importantly we have used selective membrane permeable RACK (Receptors for Activated C-Kinase) inhibitors of PKC to demonstrate that morphine-induced MOR desensitisation in LC neurones is mediated specifically by the PKCα isoform. We have also found that morphine plus PKC – induced desensitisation is absent in LC neurons from PKCα knockout mice. Thus in LC neurones PKCα mediates morphine desensitisation, whereas GRKs mediate DAMGO-induced desensitisation. It is likely that the agonists induce different conformations of MOR that, although able to couple to K⁺ current activation, nevertheless trigger different mechanisms of desensitisation. We have also developed a model for cellular tolerance to morphine, where prolonged (>6 h) exposure to morphine (30 μM) alone in LC neurons induces extensive MOR desensitisation (>80%), as quantified by operational analysis of functional receptor loss⁴. Again, PKC activity was essential to maintain morphine-induced MOR desensitisation because exposure to PKC inhibitors for only the last 30-50 min of a 6–9 h exposure to morphine reversed the MOR desensitisation. In contrast to morphine-induced desensitisation, both acute and prolonged MOR desensitisation induced by DAMGO was reduced by DNM GRK2 expression. Since PKC inhibitors can reverse morphine tolerance in vivo⁵, then it seems likely that PKC-mediated desensitisation of MORs underlies morphine tolerance. We are trying to identify the molecular mechanism(s) of PKC-mediated desensitisation of the morphine-activated MOR; initial studies indicate that PKC can phosphorylate GST fusion proteins expressing the 3rd i.c. loop and C-terminal tail of the receptor. Once the phosphorylated residues are identified, we can introduce point mutations into the intact MOR to assess their importance in morphine-induced MOR desensitisation and morphine tolerance.