␤-Arrestins serve a dual regulatory role in the life cycle of G protein-coupled receptors such as the ␤ 2 -adrenergic receptor. First, they mediate rapid desensitization by binding to G protein-coupled receptor kinasephosphorylated receptors. Second, they target the receptors for internalization into endosomal vesicles, wherein receptor dephosphorylation and resensitization occur. Here we report that phosphorylation of a carboxyl-terminal serine (Ser-412) in ␤-arrestin1 regulates its endocytotic but not its desensitization function. Cytoplasmic ␤-arrestin1 is constitutively phosphorylated and is recruited to the plasma membrane by agonist stimulation of the receptors. At the plasma membrane, ␤-arrestin1 is rapidly dephosphorylated, a process that is required for its clathrin binding and receptor endocytosis but not for its receptor binding and desensitization. Once internalized, ␤-arrestin1 is rephosphorylated. Thus, as with the classical endocytic adaptor protein complex AP2, ␤-arrestin1 functions as a clathrin adaptor in receptor endocytosis which is regulated by dephosphorylation at the plasma membrane.Endocytosis of many cell-surface receptors including those for epidermal growth factor, insulin, and transferrin is mediated by classical clathrin-coated vesicle mechanisms (1). G protein-coupled receptors such as the ␤ 2 -adrenergic receptor, M1 muscarinic cholinergic receptor, LH/HCG receptor, gastrin releasing peptide receptor, and others also utilize this pathway (2-5). In the case of tyrosine kinase receptors, endocytosis involves clustering of the receptors in coated pits formed by the recruitment and assembly of clathrin and associated molecules such as the AP2 complex and dynamin on the plasma membrane (6, 7). The heterotetrameric AP2 complex is a structural component of clathrin-coated pits on the plasma membrane that triggers the assembly of clathrin cages (8 -10). It serves as an adaptor linking receptors to the structure of clathrin cages.In the case of G protein-coupled ␤ 2 -adrenergic receptors, recent in vitro evidence has suggested that ␤-arrestins may play a role in linking the receptors to clathrin-coated pits (11-13). ␤-Arrestins were originally discovered in the context of homologous or agonist-specific desensitization of ␤ 2 -adrenergic receptors (14,15). Following phosphorylation of the agonistoccupied receptors by ␤-adrenergic receptor kinase, ␤-arrestins bind to the receptors, thereby interdicting signal transduction to heterotrimeric G proteins (16). The arrestin family includes visual arrestin, ␤-arrestin1 (arrestin2), ␤-arrestin2 (arrestin3), and other splicing variants (17). Arrestin functions specifically in inactivation of rhodopsin (18, 19), whereas ␤-arrestin1 and ␤-arrestin2 exhibit similar functions in desensitization of nonvisual G protein-coupled receptors (15).When ␤-arrestin1 or -2 are overexpressed in cells, not only is desensitization of ␤ 2 -adrenergic receptors augmented, but their sequestration or internalization is promoted as well (20). Moreover, a "dominant negative...

Acute pharmacological blockade of central histamine H 3 receptors (H 3 Rs) enhances arousal/attention in rodents. However, there is little information available for other behavioral domains or for repeated administration using selective compounds. ABT-239 [4-(2-{2-[(2R)-2-methylpyrrolidinyl]ethyl}-benzofuran-5-yl)benzonitrile] exemplifies such a selective, nonimidazole H 3 R antagonist with high affinity for rat (pK i ϭ 8.9) and human (pK i ϭ 9.5) H 3 Rs. Acute functional blockade of central H 3 Rs was demonstrated by blocking the dipsogenia response to the selective H 3 R agonist (R)-␣-methylhistamine in mice. In cognition studies, acquisition of a five-trial, inhibitory avoidance test in rat pups was improved with ABT-239 (0.1-1.0 mg/kg), a 10-to 150-fold gain in potency, with similar efficacy, over previous antagonists such as thioperamide, ciproxifan, A-304121 [(4-(3-(4 -furamide], and A-349821 [(4Ј-(3-((R,R)2,5-dimethyl-pyrrolidin-1-yl)-propoxy)-biphenyl-4-yl)-morpholin-4-yl-methanone]. Efficacy in this modelwas maintained for 3 to 6 h and following repeated dosing with ABT-239. Social memory was also improved in adult (0.01-0.3 mg/kg) and aged (0.3-1.0 mg/kg) rats. In schizophrenia models, ABT-239 improved gating deficits in DBA/2 mice using prepulse inhibition of startle (1.0 -3.0 mg/kg) and N40 (1.0 -10.0 mg/kg). Furthermore, ABT-239 (1.0 mg/kg) attenuated methamphetamineinduced hyperactivity in mice. In freely moving rat microdialysis studies, ABT-239 enhanced acetylcholine release (0.1-3.0 mg/kg) in adult rat frontal cortex and hippocampus and enhanced dopamine release in frontal cortex (3.0 mg/kg), but not striatum. In summary, broad efficacy was observed with ABT-239 across animal models such that potential clinical efficacy may extend beyond disorders such as ADHD to include Alzheimer's disease and schizophrenia.

G protein-coupled receptor kinases phosphorylate the agonist occupied conformation of G protein-coupled receptors in the plasma membrane, leading to their desensitization. Receptor resensitization requires receptor dephosphorylation, a process which is mediated by a plasma and vesicular membrane-associated form of PP-2A. We present evidence that, like receptor phosphorylation, receptor dephosphorylation is tightly regulated, requiring a specific receptor conformation induced by vesicular acidification. In vitro, spontaneous dephosphorylation of phosphorylated receptors is observed only at acidic pH. Furthermore, in intact cells upon agonist stimulation, phosphorylated receptors traffic from the plasma membrane to vesicles where they become physically associated with the phosphatase and dephosphorylated. Treatment of cells with NH 4 Cl, which disrupts the acidic pH found in endosomal vesicles, blocks association of the receptors with the phosphatase and blocks receptor dephosphorylation. These findings suggest that a conformational change in the receptor induced by acidification of the endosomal vesicles is the key determinant regulating receptor dephosphorylation and resensitization.