Background: Arrestin mediates G protein-independent signaling and internalization of the D 2 receptor. Results: A D 2 receptor mutant with modestly diminished ability to recruit arrestin and ␤2-adaptin did not internalize in response to agonists. Conclusion: Arrestin-mediated recruitment of receptor to AP2 is not sufficient for internalization. Significance: Receptor mutants lacking specific functions are tools for analysis of signaling mechanisms.

Prior studies showed that tyrosine 12 phosphorylation in the N-terminal, cytoplasmic domain of the G-protein-gated inwardly rectifying potassium channel, K ir 3.1 facilitates channel deactivation by increasing intrinsic GTPase activity of the channel. Using a phosphoselective antibody directed against this residue (pY12), we now report that partial sciatic nerve ligation increased pY12-K ir 3.1-immunoreactivity (ir) in the ipsilateral dorsal horn of wildtype mice, but not in mice lacking the -opioid receptor (KOR) or lacking the G-protein receptor kinase 3 (GRK3) genes. Treatment of AtT-20 cells stably expressing KOR-GFP with the selective KOR agonist U50,488 increased both phospho-p38-ir and pY12-K ir 3.1-ir. The U50,488-induced increase in pY12-K ir 3.1-ir was blocked by the p38 inhibitor SB203580. Cells expressing KOR(S369A)-GFP did not increase either phospho-p38-ir or pY12-K ir 3.1-ir following U50,488 treatment. Whole cell voltage clamp of AtT-20 cells expressing KOR-GFP demonstrated that p38 activation by U50,488 reduced somatostatin-evoked K ir 3 currents. This heterologous desensitization was blocked by SB203580 and was not evident in cells expressing KOR(S369A)-GFP. Tyrosine phosphorylation of K ir 3.1 was likely mediated by p38 MAPK activation of Src kinase. U50,488 also increased (pY418)Src-ir; this increase was blocked by SB203580 and not evident in KOR(S369A)-GFP expressing AtT20 cells; the Src inhibitor PP2 blocked the U50,488-induced increase in pY12-K ir 3.1-ir; and the heterologous desensitization of K ir 3 currents was blocked by PP2. These results suggest that KOR causes phosphorylation of Y12-K ir 3.1 and channel inhibition through a GRK3-, p38 MAPK-and Src-dependent mechanism. Reduced inward potassium current following nerve ligation would increase dorsal horn neuronal excitability and may contribute to the neuropathic pain response.

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The effects of phosphorylation of the tyrosine residue in the highly conserved DRY motif expressed in the putative second cytoplasmic loop of the -opioid receptor were assessed after expression in human embryonic kidney (HEK) 293 cells. Tyrosine kinase activation by epidermal growth factor (EGF) or hydrogen peroxide treatment effectively increased phosphorylation of the tyrosine-166 in the -opioid receptor (MORTyr166p) as measured by a novel phosphoselective antibody. We were surprised to find that the increase in MOR-Tyr166p immunoreactivity (ir) required coactivation by the opioid agonist [D-Ala 2 ,methyl-Phe 4 ,Gly 5 -ol]enkephalin (DAMGO), as demonstrated by both Western blot imaging of membrane proteins and confocal microscopy of transfected cells; MOR-Tyr166p-ir did not significantly increase after either DAMGO, EGF, or H 2 O 2 treatment alone. The increase in MOR-Tyr166p-ir was blocked by pretreatment with the opioid antagonist naloxone or the Src kinase inhibitor 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo [3,4-d]pyrimidine. Consistent with these data, mutation of the tyrosine-166 to phenylalanine blocked the increased immunoreactivity, and untransfected HEK293 cells did not increase MOR-Tyr166p-ir after treatment. DAMGO The -opioid receptor (MOR; OPRM1) belongs to the class A (rhodopsin family) G i/o -coupled family of G-protein-coupled receptors (GPCRs) and functions to reduce neuronal excitability primarily by increasing potassium conductance and inhibiting voltage-gated calcium channels (Law et al., 2000;Williams et al., 2001). The opioid system is usually described within the context of drug abuse and analgesic drug action; however, the normal physiological role of the opioid system is to regulate pain sensitivity, endocrine functioning, gut motility, and smooth muscle tone in response to physiological stressors (López et al., 1999;Drolet et al., 2001). The regulation of -opioid signaling is a dynamic and complex process (Law et al., 2000). A primary desensitization mechanism involving G-protein receptor kinase (GRK) and ␤-arrestindependent internalization through cytoplasmic serine/threonine phosphorylation has been well described previously (Celver et al., 2001(Celver et al., , 2004Williams et al., 2001). In addition, MOR contains four highly conserved cytoplasmic tyrosine residues (Thompson et al., 1993), and tyrosine kinase-mediated mechanisms regulating MOR signaling have also been described previously (McLaughlin and Chavkin, 2001;Zhang et al., 2009). Tyrosine phosphorylation may influence MOR trafficking and signaling (Pak et al., 1999), consistent with the effects of tyrosine phosphorylation on internalization and signaling of the ␦-and -opioid receptors (Kramer et al., 2000;Appleyard et al., 2000). A recent study by Law and colleagues showed that tyrosine phosphorylation of MOR at Tyr166 and Tyr336 controlled the switch from inhibition to

The periaqueductal gray (PAG) and rostral ventromedial medulla (RVM) are part of a nociceptive modulatory system. Microinjection of morphine into either structure produces antinociception. Tolerance develops to ventrolateral PAG mediated antinociception with repeated microinjection of morphine. In contrast, there are no published reports of tolerance to morphine administration into the RVM. Three experiments were conducted to determine whether tolerance develops to morphine microinjections into the RVM. Experiment 1 compared tolerance to the antinociceptive effect of microinjecting morphine (5 microg/0.5 microl) into the PAG and RVM following daily injections for four consecutive days. Experiment 2 assessed tolerance to a range of morphine doses (2.5-20 microg) after injecting morphine into the RVM twice a day for two consecutive days. Experiment 3 followed a similar procedure except twice as many RVM injections were made (8 microinjections in 4 days). The degree to which tolerance developed to the antinociceptive effect of morphine was much greater with microinjections into the PAG compared to the RVM. There was a 64% drop in hot plate latency from the first to the fifth injection of morphine into the PAG, but only a 36% drop in latency following RVM microinjections. Reducing the interdose interval to two injections a day or increasing the total number of injections from 4 to 8 did not enhance the development of tolerance to RVM morphine administration. These data demonstrate that opioid-sensitive neurons in the RVM are relatively resistant to the development of tolerance compared to PAG neurons.