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Open Access Publications from the University of California

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

  • Author(s): Bhatnagar, A
  • Willins, DL
  • Gray, JA
  • Woods, J
  • Benovic, JL
  • Roth, BL
  • et al.

5-Hydroxytryptamine 2A (5-HT2A) receptors, a major site of action of clozapine and other atypical antipsychotic medications, are, paradoxically, internalized in vitro and in vivo by antagonists and agonists. The mechanisms responsible for this paradoxical regulation of 5-HT2Areceptors are unknown. In this study, the arrestin and dynamin dependences of agonist- and antagonist-mediated internalization were investigated in live cells using green fluorescent protein (GFP)-tagged 5-HT2Areceptors (SR2-GFP). Preliminary experiments indicated that GFP tagging of 5-HT2Areceptors had no effect on either the binding affinities of several ligands or agonist efficacy. Likewise, both the native receptor and SR2-GFP were internalized via endosomes in vitro. Experiments with a dynamin dominant-negative mutant (dynamin K44A) demonstrated that both agonist- and antagonist-induced internalization were dynamin-dependent. By contrast, both the agonist- and antagonist-induced internalization 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-HT2Areceptor activation by agonists, but not antagonists, induced greater Arr-3 than Arr-2 translocation to the plasma membrane. Importantly, the agonist-induced internalization of 5-HT2Areceptors was accompanied by differential sorting of Arr-2, Arr-3, and 5-HT2Areceptors into distinct plasma membrane and intracellular compartments. The agonist-induced redistribution of Arr-2 and Arr-3 into intracellular vesicles and plasma membrane compartments distinct from those involved in 5-HT2Areceptor internalization implies novel roles for Arr-2 and Arr-3 independent of 5-HT2Areceptor internalization and desensitization.

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