The dynamin-dependent, arrestin-independent internalization of 5-hydroxytryptamine 2A (5-HT2A) serotonin receptors reveals differential sorting of arrestins and 5-HT2A receptors during endocytosis

5-Hydroxytryptamine 2A (5-HT2A) receptors, a major site of action of clozap ine and other atypical antipsychotic medications, are, paradoxically, inter nalized in vitro and in vivo by antagonists and agonists, The mechanisms re sponsible for this paradoxical regulation of 5-HT2A receptors are unknown. In this study, the arrestin and dynamin dependences of agonist- and antagon ist-mediated internalization were investigated in live cells using green fl uorescent protein (GFP)-tagged 5-HT2A receptors (SR2-GFP). Preliminary expe riments indicated that GFP tagging of 5-HT2A receptors had no effect on eit her the binding affinities of several ligands or agonist efficacy. Likewise , both the native receptor and SR2-GFP were Internalized via endosomes in v itro, Experiments with a dynamin dominant-negative mutant (dynamin K44A) de monstrated that both agonist- and antagonist-induced internalization were d ynamin-dependent. By contrast, both the agonist- and antagonist-induced int ernalization of SR2-GFP were insensitive to three different arrestin (Arr) dominant-negative mutants (Arr-2 V53D, Arr-2-(319-418), and Arr-3-(284-409) ), Interestingly, 5-HT2A receptor activation by agonists, but not antagonis ts, induced greater Arr-3 than Arr-2 translocation to the plasma membrane. Importantly, the agonist-induced internalization of 5-HT2A receptors was ac companied by differential sorting of Arr-2, Arr-3, and 5-HT2A receptors int o distinct plasma membrane and intracellular compartments. The agonist-indu ced redistribution of Arr-2 and Arr-3 into intracellular vesicles and plasm a membrane compartments distinct from those involved in 5-HT2A receptor int ernalization implies novel roles for Arr-2 and Arr-3 independent of 5-HT2A receptor internalization and desensitization.