Previously, we reported that the time course for the rapid phosphorylation rate of mu-opioid receptor expressed in human embryonic kidney (HEK)293 cells did not correlate with the slow receptor desensitization rate induced by [D-Ala(2),N-MePhe(4), Gly-ol(5)]-enkephalin (DAMGO). However, others have suggested that receptor phosphorylation is the trigger for mu-opioid receptor desensitization. In this study, we demonstrated the relatively slow rate of receptor desensitization could be attributed partially to the recycling of internalized receptor as determined by fluorescence-activated cell-sorting analysis. However, the blockade of the endocytic and Golgi transport events in HEK293 cells with monensin and brefeldin A did not increase the initial rate of receptor desensitization. But the desensitization rate was increased by reduction of the mu-opioid receptor level with beta-furnaltrexamine (betaFNA). The reduction of the receptor level with 1 microM betaFNA significantly increased the rate of etorphine-induced receptor desensitization. By blocking the ability of receptor to internalize with 0.4 M sucrose, a significant degree of receptor being rapidly desensitized was observed in HEK293 cells pretreated with betaFNA. Hence, mu-opioid receptor is being resensitized during chronic agonist treatment. The significance of resensitization of the internalized receptor in affecting receptor desensitization was demonstrated further with human neuroblastoma SHSY5Y cells that expressed a low level of mu-opioid receptor. Although DAMGO could not induce a rapid desensitization in these cells, in the presence of monensin and brefeldin A, DAMGO desensitized the mu-opioid receptor's ability to regulate adenylyl cyclase with a t(1/2) = 9.9 +/- 2.1 min and a maximal desensitized level at 70 +/- 4.7%. Furthermore, blockade of receptor internalization with 0.4 M sucrose enhanced the DAMGO-induced receptor desensitization, and the inclusion of monensin prevented the resensitization of the mu-opioid receptor after chronic agonist treatment in SHSY5Y cells. Thus, the ability of the mu-opioid receptor to resensitize and to recycle, and the relative efficiency of the receptor to regulate adenylyl cyclase activity, contributed to the observed slow rate of mu-opioid receptor desensitization in HEK293 cells.

The discovery of a series of pyrrole-sulfonamides as positive allosteric modulators (PAM) of alpha7 nAChRs is described. Optimization of this series led to the identification of 19 (A-867744), a novel type II PAM with good potency and selectivity. Compound 19 showed acceptable pharmacokinetic profile across species and brain levels sufficient to modulate alpha7 nAChRs. In a rodent model of sensory gating, 19 normalized gating deficits. These results suggest that 19 represents a novel class of molecules capable of allosteric modulation of the alpha7 nAChRs.

Agonist-induced internalization of G protein-coupled receptors is influenced by many structural determinants including the carboxyl tail. To investigate the role of serine and threonine residues within the carboxyl tail, several mutants were constructed by truncating the carboxyl tail of the hemagglutinin-tagged -opioid receptor, thereby removing serines and threonines systematically. Neuro 2A cells stably expressing the truncated receptors did not exhibit a significant alteration in the affinity of Prolonged exposure to opioid agonists results in a loss of effector response termed desensitization. It is thought that the overall desensitization of opioid receptors follows that of other G-protein-coupled receptors (GPCR), 1 where cellular adaptations result in conformational changes that uncouple the receptors from their respective G-proteins. Such molecular changes also lead to an ultimate loss of receptors from the cell surface via internalization and degradation pathways. The mechanisms and specific amino acids which facilitate these processes, however, have not been delineated.While nearly all GPCRs follow this overall process of desensitization, the ␤-adrenergic receptor has served as a model for characterizing such mechanisms. In the ␤-adrenergic receptor, agonist-activation initiates receptor phosphorylation by multiple kinases including protein kinase C (PKC), protein kinase A (PKA), and G-protein-coupled receptor kinases (GRK). Phosphorylation of the receptor, in turn, enhances the affinity of ␤-arrestin, an associative protein, which advances receptor internalization leading to receptor degradation (1). This paradigm of receptor phosphorylation has been shown to account for the agonist-induced receptor internalization of numerous other members of the GPCR family (2-6). There is, however, no consensus among the GPCR family as to a common motif for down-regulation, but phosphorylation of the receptor appears to be a prerequisite.Studies have indicated that opioid receptors are rapidly phosphorylated after agonist activation as well. Zhang and colleagues (7) demonstrated that both morphine and phorbol 12-myristate 13-acetate, a PKC activator, stimulated phosphorylation of the -opioid receptor expressed in Chinese hamster ovary cells within 5 min of exposure. They then suggested a correlation between the time course of this phosphorylation event with that of the desensitization of the inwardly rectifying potassium channel (GIRK) to opioid agonists in Xenopus oocytes expressing both the -opioid receptor and channel (7). These studies in the oocytes were supported by a study with Chinese hamster ovary cells stably expressing the wild-type and mutant receptors when Pak and colleagues (8) reported that point mutation of Thr 394 to alanine completely blocked desensitization of the receptor to DAMGO after just 1 h of exposure. They proposed that phosphorylation of this specific threonine individually gated the desensitization of the -opioid receptor. However, studies by Kovoor and co-workers (9) directly contradi...

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