AtT-20 cells expressing the wild-type kappa opioid receptor (KOR) increased phospho-p38 MAPK following treatment with the kappa agonist U50,488. The increase was blocked by the kappa antagonist norbinaltorphimine and not evident in untransfected cells. In contrast, U50,488 treatment of AtT-20 cells expressing KOR having alanine substituted for serine-369 (KSA) did not increase phospho-p38. Phosphorylation of serine 369 in the KOR carboxyl terminus by G-protein receptor kinase 3 (GRK3) was previously shown to be required for receptor desensitization, and the results suggest that p38 MAPK activation by KOR may require arrestin recruitment. This hypothesis was tested by transfecting arrestin3-(R170E), a dominant positive form of arrestin that does not require receptor phosphorylation for activation. AtT-20 cells expressing both KSA and arrestin3-(R170E) responded to U50,488 treatment with an increase in phospho-p38 consistent with the hypothesis. Primary cultured astrocytes (glial fibrillary acidic protein-positive) and neurons (␥-aminobutyric acid-positive) isolated from mouse striata also responded to U50,488 by increasing phospho-p38 immunolabeling. p38 activation was not evident in either striatal astrocytes or neurons isolated from KOR knock-out mice or GRK3 knock-out mice. Astrocytes pretreated with small interfering RNA for arrestin3 were also unable to activate p38 in response to U50,488 treatment. Furthermore, in striatal neurons, the kappamediated phospho-p38 labeling was colocalized with arrestin3. These findings suggest that KOR may activate p38 MAPK in brain by a GRK3 and arrestin-dependent mechanism.Kappa opioid receptors are G-protein-coupled receptors (GPCRs) 2 that are widely expressed throughout the brain and are activated by the endogenous opioid peptides derived from prodynorphin (1, 2). Several reports have characterized the signal transduction events initiated by KOR activation. By coupling to the G-protein G␣ i/o , KOR inhibits adenylate cyclase, increases potassium conductance, decreases calcium conductance, and mobilizes intracellular calcium (3). More recently, KOR has been recognized to activate the extracellular signalregulated kinase (ERK 1/2) (4, 5). This activation has been demonstrated to persist for several hours following agonist treatment, suggesting a role for KOR in long term growth and gene regulation. Other studies have demonstrated that KOR can activate c-Jun amino-terminal kinase (6). These studies suggest that KOR can activate multiple signaling pathways that result in the immediate and long term cellular effects of kappa opioids.Sustained agonist exposure causes GPCR phosphorylation and desensitization (7). For the kappa opioid receptor, G-protein receptor kinase 3 (GRK3) phosphorylation of serine 369 in the carboxyl-terminal domain of KOR initiates arrestin-dependent receptor desensitization and internalization (8, 9). Recently, new evidence suggests that the arrestin-bound GPCR is not inactive and instead can recruit MAPK signaling modules (10). For example, for the chem...

Key points• Dopamine's control over excitatory signals from the cortex to the nucleus accumbens is thought to underlie motor learning, behavioural reinforcement and drug dependence.• In this study, we combined optical recordings of presynaptic release with whole-cell electrophysiology in CB 1 receptor-null mice and bacterial artificial chromosome (BAC) transgenic mice with fluorescently labelled D1 and D2 receptor-expressing neurons to identify the specific interactions between dopamine and glutamate signalling at individual cortical terminals within the nucleus accumbens core.• Experiments showed that dopamine produces frequency-dependent filtering of low-probability release synapses. At low frequencies, D1 receptors excited striatal output neurons of the striatonigral and striatopallidal pathways, while D2 receptors specifically inhibited neurons of the striatopallidal pathway. At higher frequencies, the dopamine-dependent release of adenosine and endocannabinoids promoted further temporal filtering of cortical signals entering both output pathways.• These results help us understand how dopamine provides frequency and temporal filtering of cortical information by promoting activity through the striatonigral pathway, while inhibiting weak signals.Abstract Interactions between dopamine and glutamate signalling within the nucleus accumbens core are required for behavioural reinforcement and habit formation. Dopamine modulates excitatory glutamatergic signals from the prefrontal cortex, but the precise mechanism has not been identified. We combined optical and electrophysiology recordings in murine slice preparations from CB 1 receptor-null mice and green fluorescent protein hemizygotic bacterial artificial chromosome transgenic mice to show how dopamine regulates glutamatergic synapses specific to the striatonigral and striatopallidal basal ganglia pathways. At low cortical frequencies, dopamine D1 receptors promote glutamate release to both D1 and D2 receptor-expressing medium spiny neurons while D2 receptors specifically inhibit excitatory inputs to D2 receptor-expressing cells by decreasing exocytosis from cortical terminals with a low probability of release. At higher cortical stimulation frequencies, this dopaminergic modulation of presynaptic activity is occluded by adenosine and endocannabinoids. Glutamatergic inputs to both D1 and D2 receptor-bearing medium spiny neurons are inhibited by adenosine, released upon activation of NMDA and AMPA receptors and adenylyl cyclase in D1 receptor-expressing cells. Excitatory inputs to D2 receptor-expressing cells are specifically inhibited by endocannabinoids, whose release is dependent on D2 and group 1 metabotropic glutamate receptors. The convergence of excitatory and inhibitory modulation of corticoaccumbal activity by dopamine, adenosine and endocannabinoids creates subsets of corticoaccumbal inputs, selectively and temporally reinforces strong cortical signals through the striatonigral pathway while inhibiting the weak, and may provide a mechanism whereby conti...

, arr2(R169E), that desensitizes G protein-coupled receptors in an agonist-dependent but phosphorylation-independent manner. arr2(R169E) produced robust desensitization of MOR and MOR-(T180A) in the absence of GRK3 coexpression. These results demonstrate that the T180A mutation probably blocks GRK3-and arr3-mediated desensitization of MOR by preventing a critical agonist-dependent receptor phosphorylation and suggest a novel GRK3 site of regulation not yet described for other G-protein-coupled receptors.

Homologous desensitization of the opioid receptor (OR) can be resolved into distinct processes that include the uncoupling of the OR from its G-protein effectors and internalization of cell surface receptors. Using electrophysiological recordings of OR activation of G-protein-coupled K ϩ channels (K ir 3) in Xenopus laevis oocytes and AtT20 cells, confocal microscopy of receptor localization, and radioligand binding of cell surface receptors, we resolved these desensitization mechanisms to determine the domain of OR important for receptor uncoupling. Activation of OR by saturating concentrations of [D-Ala 2 ,N-Me-Phe 4 ,Gly 5 -ol]-enkephalin (DAMGO), methadone, or fentanyl, but not morphine, produced robust internalization of a green fluorescent protein-tagged OR. A subsaturating concentration of DAMGO (100 nM) did not cause receptor internalization but markedly reduced the subsequent responsiveness of K ir 3 by uncoupling OR. OR desensitization in AtT20 cells was confirmed to be homologous, because desensitization by 100 nM DAMGO was blocked by dominant-negative forms of either G protein-coupled receptor kinase (GRK) or arrestin, and pretreatment with DAMGO did not affect the K ir 3 response to somatostatin receptor activation. Alanine substitution of a single threonine in the second cytoplasmic loop of the OR (Threonine 180) blocked agonist-dependent receptor uncoupling without affecting receptor internalization. These results suggest that GRK-dependent phosphorylation of OR required threonine 180 for uncoupling but that a different GRK and arrestin-dependent mechanism controlled OR internalization in AtT20 cells.

1 Tolerance to opioids frequently follows repeated drug administration and affects the clinical utility of these analgesics. Studies in simple cellular systems have demonstrated that prolonged activation of opioid receptors produces homologous receptor desensitization by G-protein receptor kinase mediated receptor phosphorylation and subsequent b-arrestin binding. To define the role of this regulatory mechanism in the control of the electrophysiological and behavioral responses to opioids, we used mice having a targeted disruption of the G-protein receptor kinase 3 (GRK3) gene. 2 Mice lacking GRK3 did not differ from wild-type littermates neither in their response latencies to noxious stimuli on the hot-plate test nor in their acute antinociceptive responses to fentanyl or morphine. 3 Tolerance to the electrophysiological response to the opioid fentanyl, measured in vitro in the hippocampus, was blocked by GRK3 deletion. In addition, tolerance to the antinociceptive effects of fentanyl was significantly reduced in GRK3 knockouts compared to wild-type littermate controls. 4 Tolerance to the antinociceptive effects of morphine was not affected by GRK3 deletion although morphine tolerance in hippocampal slices from GRK3 knockout mice was significantly inhibited. Tolerance developed more slowly in vitro to morphine than fentanyl supporting previous work in in vitro systems showing a correlation between agonist efficacy and GRK3-mediated desensitization. 5 The results of these studies suggest that GRK3-mediated mechanisms are important components of both electrophysiologic and behavioral opioid tolerance. Fentanyl, a high efficacy opioid, more effectively produced GRK3-dependent effects than morphine, a low efficacy agonist.